Identification of Secondary Prevention Patients Eligible for PCSK9 Inhibitors Therapy According to the Routine Clinical Practice in Spain.

INTRODUCTION: Many patients at very high risk of cardiovascular (CV) events would benefit from lipid-lowering therapies (LLT) intensification to decrease their risk. This study aimed to identify the real-world secondary prevention patients potentially eligible for proprotein convertase subtilisin-kexin type 9 inhibitors (PCSK9i) in Spain. METHODS: This retrospective cohort study included adult patients registered in the IQVIA Spanish Electronic Medical Records outpatient database (2014-2020), diagnosed with myocardial infarction (MI), unstable angina (UA), ischaemic stroke (IS), transient ischaemic attack (TIA) or peripheral artery disease (PAD) and with ≥ 1 low-density lipoprotein cholesterol (LDL-C) or total cholesterol measurements. Longitudinal data were collected from the initial diagnosis to the end of the study period or follow-up loss. RESULTS: The study included 9516 patients, 63.9% male, mean (SD) age 67.7 (12.5) years and mean LDL-C 117.3 (38.8) mg/dL. MI, IS and PAD were the most severe events reported during the study period (28.5%, 18.7% and 29.3% of patients, respectively). At the time of last available LDL-C assessment (≥ 3 months post-event), 64.4% patients were on LLT. Of those, 45.4%, 46.9% and 7.7% were on high-, moderate- and low-intensity LLT. Overall, 9.6% patients achieved LDL-C < 55 mg/dL (24.2% LDL-C < 70 mg/dL). Furthermore, 17.9% patients receiving optimized oral LLT showed LDL-C > 100 mg/dL (LDL-C reimbursement threshold for PCSK9i in Spain). CONCLUSION: Up to 82% of patients with atherosclerotic CV disease do not achieve LDL-C levels recommended by the 2019 ESC/EAS guidelines despite being on optimized oral LLT therapy. In 17.9% of these patients LDL-C levels exceed 100 mg/dL, being eligible for PCSK9i in Spain.

Manejo quirúrgico del paciente con hemofilia sometido a cirugía bucal: Reporte de un caso clínico / Surgical management of the patient with hemophilia undergoing oral surgery: A case report

Resumen Introducción: La hemofilia es una deficiencia congénita de un factor de la coagulación, la cual consta en un trastorno recesivo asociado al cromosoma X, generando disminución o ausencia de actividad funcional del factor. Objetivo: Presentar una revisión narrativa de la literatura sobre pacientes hemofílicos, junto con un caso de manejo de un paciente con la condición. Metodología: Paciente de sexo femenino, 18 años, acude al postgrado de Cirugía y Traumatología bucomaxilofacial de la Universidad Andrés Bello de Santiago de Chile, derivada para realizar exodoncia de terceros molares debido al término de su mecánica ortodóntica. Al realizar la anamnesis próxima, la paciente relata padecer hemofilia A leve, y hace 6 meses presentó un 38% de factor VIII. Previo al tratamiento quirúrgico se solicitó un hemograma completo con examen de coagulación para medir el TTPA. Además, se realizó una interconsulta con el hematólogo tratante para evaluación de su patología y recomendaciones para efectuar la misma con la menor cantidad de riesgos intraquirúrgicos y postquirúrgicos, el cual sugirió la administración de factor VIII previo, y posterior al acto quirúrgico. Así mismo, se aplicaron medidas de hemostasia locales para mejor control y un correcto manejo analgésico postquirúrgico. Conclusión: La hemofilia, es un trastorno que requiere un minucioso manejo tanto pre, intra y postoperatorio de parte del odontólogo, donde los exámenes complementarios, comunicación con el hematólogo, procedimiento atraumático y un correcto manejo de la hemostasia, son fundamentales para el éxito del tratamiento.

Abstract Introduction: Hemophilia is a congenital deficiency of a coagulation factor, associated to a recessive pattern located in the X chromosome, which induces a lower or even absent functional activity of that factor. Objective: To provide a narrative review of the literature about haemophiliac patients, as well as a case report of a patient. Methods: Female patient, 18 years old, attended in the postgraduate of Maxillofacial Surgery of the Andrés Bello University to Santiago, Chile, derived to perform extractions of wisdom teeth due to the end of its orthodontic mechanics. At the anamnesis, the patient reports to suffer from mild hemophilia A, and 6 months ago she had 38% VIII factor. Prior to surgical treatment, a complete blood count with a coagulation test was requested to measure TTPA. In addition, an interconsultation was made with the treating hematologist to perform a correct management to assess of her pathology and recommendations to carry out it with the least amount of intrasurgical and post-surgical risks. Suggested the administration of factor of freeze-dried VIII factor before and after surgery. Local hemostasis measures were also applied for better control and proper post-surgical pain management. Conclusion: Hemophilia, requires the dentist to perform a thorough management pre, intra and postoperatory, in which complementary tests, communication with the hematologist, atraumatic procedure and a precise management of hemostasis, are key for the treatment's success.

DRUG EVALUATION AND DECISION MAKING IN CATALONIA: DEVELOPMENT AND VALIDATION OF A METHODOLOGICAL FRAMEWORK BASED ON MULTI-CRITERIA DECISION ANALYSIS (MCDA) FOR ORPHAN DRUGS.

OBJECTIVES: The aim of this study was to adapt and assess the value of a Multi-Criteria Decision Analysis (MCDA) framework (EVIDEM) for the evaluation of Orphan drugs in Catalonia (Catalan Health Service). METHODS: The standard evaluation and decision-making procedures of CatSalut were compared with the EVIDEM methodology and contents. The EVIDEM framework was adapted to the Catalan context, focusing on the evaluation of Orphan drugs (PASFTAC program), during a Workshop with sixteen PASFTAC members. The criteria weighting was done using two different techniques (nonhierarchical and hierarchical). Reliability was assessed by re-test. RESULTS: The EVIDEM framework and methodology was found useful and feasible for Orphan drugs evaluation and decision making in Catalonia. All the criteria considered for the development of the CatSalut Technical Reports and decision making were considered in the framework. Nevertheless, the framework could improve the reporting of some of these criteria (i.e., "unmet needs" or "nonmedical costs"). Some Contextual criteria were removed (i.e., "Mandate and scope of healthcare system", "Environmental impact") or adapted ("population priorities and access") for CatSalut purposes. Independently of the weighting technique considered, the most important evaluation criteria identified for orphan drugs were: "disease severity", "unmet needs" and "comparative effectiveness", while the "size of the population" had the lowest relevance for decision making. Test-retest analysis showed weight consistency among techniques, supporting reliability overtime. CONCLUSIONS: MCDA (EVIDEM framework) could be a useful tool to complement the current evaluation methods of CatSalut, contributing to standardization and pragmatism, providing a method to tackle ethical dilemmas and facilitating discussions related to decision making.

Manejo quirúrgico del paciente con hemofilia sometido a cirugía bucal: Reporte de un caso clínico

Introducción: La hemofilia es una deficiencia congénita de un factor de la coagulación, la cual consta en un trastorno recesivo asociado al cromosoma X, generando disminución o ausencia de actividad funcional del factor. Objetivo: Presentar una revisión narrativa de la literatura sobre pacientes hemofílicos, junto con un caso de manejo de un paciente con la condición. Metodología: Paciente de sexo femenino, 18 años, acude al postgrado de Cirugía y Traumatología bucomaxilofacial de la Universidad Andrés Bello de Santiago de Chile, derivada para realizar exodoncia de terceros molares debido al término de su mecánica ortodóntica. Al realizar la anamnesis próxima, la paciente relata padecer hemofilia A leve, y hace 6 meses presentó un 38% de factor VIII. Previo al tratamiento quirúrgico se solicitó un hemograma completo con examen de coagulación para medir el TTPA. Además, se realizó una interconsulta con el hematólogo tratante para evaluación de su patología y recomendaciones para efectuar la misma con la menor cantidad de riesgos intraquirúrgicos y postquirúrgicos, el cual sugirió la administración de factor VIII previo, y posterior al acto quirúrgico. Así mismo, se aplicaron medidas de hemostasia locales para mejor control y un correcto manejo analgésico postquirúrgico. Conclusión: La hemofilia, es un trastorno que requiere un minucioso manejo tanto pre, intra y postoperatorio de parte del odontólogo, donde los exámenes complementarios, comunicación con el hematólogo, procedimiento atraumático y un correcto manejo de la hemostasia, son fundamentales para el éxito del tratamiento.

Introduction: Hemophilia is a congenital deficiency of a coagulation factor, associated to a recessive pattern located in the X chromosome, which induces a lower or even absent functional activity of that factor. Objective: To provide a narrative review of the literature about haemophiliac patients, as well as a case report of a patient. Methods: Female patient, 18 years old, attended in the postgraduate of Maxillofacial Surgery of the Andrés Bello University to Santiago, Chile, derived to perform extractions of wisdom teeth due to the end of its orthodontic mechanics. At the anamnesis, the patient reports to suffer from mild hemophilia A, and 6 months ago she had 38% VIII factor. Prior to surgical treatment, a complete blood count with a coagulation test was requested to measure TTPA. In addition, an interconsultation was made with the treating hematologist to perform a correct management to assess of her pathology and recommendations to carry out it with the least amount of intrasurgical and post-surgical risks. Suggested the administration of factor of freeze-dried VIII factor before and after surgery. Local hemostasis measures were also applied for better control and proper post-surgical pain management. Conclusion: Hemophilia, requires the dentist to perform a thorough management pre, intra and postoperatory, in which complementary tests, communication with the hematologist, atraumatic procedure and a precise management of hemostasis, are key for the treatment's success.

Does Combined Use of the J-tip® and Buzzy® Device Decrease the Pain of Venipuncture in a Pediatric Population?

BACKGROUND: Both the J-tip® (a needle-free device for subcutaneous delivery of lidocaine) and the Buzzy® (a cooled, vibrating device to employ gate control to minimize procedural pain) have shown some efficacy in diminishing the pain of venipuncture. PURPOSE: To develop an optimal protocol for pre-venipuncture/IV start pain management by investigating the impact of adding the use of Buzzy® prior to the use of the J-tip®. PROCEDURES: Pediatric emergency department patients aged 1 month to 21 years were prospectively enrolled in Phase 1 (J-tip® only) then Phase 2 (Buzzy®+J-tip®) for analgesia prior to venipuncture or IV start. Age-appropriate pain scale scores were collected for the subsequent procedure, as well the administration of lidocaine via J-tip®. MAIN FINDINGS: With the combined intervention (phase 2), 14.2% of patients had a pain scale score >3 with venipuncture and 16.1% had a pain scale score >3 with application of the J-tip® itself. With no intervention for pain relief, 71% of patients experienced a pain scale score >3 for venipuncture. With the J-tip® alone (phase 1), 21% had a pain scale score >3 with venipuncture and 22.3% had a pain scale score >3 with application of the J-tip® itself. CONCLUSIONS: Patients receiving either intervention reported lower scores on pain scales during venipuncture or IV start than the no analgesia group. The combined intervention did not yield a significant decrease in scores on pain scale scores over the J-tip® alone.

Metabolomics discloses donor liver biomarkers associated with early allograft dysfunction.

BACKGROUND & AIMS: Early allograft dysfunction (EAD) dramatically influences graft and patient outcome after orthotopic liver transplantation and its incidence is strongly determined by donor liver quality. Nevertheless, objective biomarkers, which can assess graft quality and anticipate organ function, are still lacking. This study aims to investigate whether there is a preoperative donor liver metabolomic biosignature associated with EAD. METHODS: A comprehensive metabolomic profiling of 124 donor liver biopsies collected before transplantation was performed by mass spectrometry coupled to liquid chromatography. Donor liver grafts were classified into two groups: showing EAD and immediate graft function (IGF). Multivariate data analysis was used to search for the relationship between the metabolomic profiles present in donor livers before transplantation and their function in recipients. RESULTS: A set of liver graft dysfunction-associated biomarkers was identified. Key changes include significantly increased levels of bile acids, lysophospholipids, phospholipids, sphingomyelins and histidine metabolism products, all suggestive of disrupted lipid homeostasis and altered histidine pathway. Based on these biomarkers, a predictive EAD model was built and further evaluated by assessing 24 independent donor livers, yielding 91% sensitivity and 82% specificity. The model was also successfully challenged by evaluating donor livers showing primary non-function (n=4). CONCLUSIONS: A metabolomic biosignature that accurately differentiates donor livers, which later showed EAD or IGF, has been deciphered. The remarkable metabolomic differences between donor livers before transplant can relate to their different quality. The proposed metabolomic approach may become a clinical tool for donor liver quality assessment and for anticipating graft function before transplant.

Cost of clinical management of acromegaly in Spain.

INTRODUCTION AND BACKGROUND: The cost of the therapeutic management of acromegaly depends on the selection of resources used, surgery and/or pharmacological treatment, by the specialist responsible for treatment, related to the characteristics of the patient and tumour. The objective of this work is to evaluate these costs for an illness that is rare but that is associated with a high morbidity in the context of routine clinical practice. METHODS: This was an epidemiological, prospective, naturalistic, multicentre study in Spain, in which 38 endocrinologists participated. Adult patients with acromegaly and a pituitary microadenoma or macroadenoma were included in the study. Patients were assigned, according to first-line treatment, to the following two groups: surgery first-line group (surgery in the 6 months before inclusion or during the follow-up period) and pharmaceutical first-line group (treatment with somatostatin analogues [SAs] for at least 6 months and with or without surgery after starting treatment with SAs). Data were collected during routine visits made during a follow-up period of 2 years. All resources were estimated at 2009 prices (€) and adjusted according to the Spanish consumer price index in 2010. RESULTS: Seventy-four patients were included, the majority of them with macroadenoma (70%). Eighty-eight percent of patients were treated surgically (76% as a first-line treatment), while 12% of patients received only SAs. Treatment with SAs was used at some point in the study by 85% of patients. The mean annual total cost of acromegaly is €9668 per patient (€9223 for the surgery group and €11,054 for the pharmaceutical group). Seventy-one percent of the direct cost of the disease corresponds to treatment with SAs. The cost of a patient treated only with surgery is €2501 on an annual basis, versus €9745 for a patient receiving only pharmacological treatment. In cases where a combination of both types of treatment is required, the annual total cost ranges from €10,866 to €12,364. CONCLUSION: The annual direct cost per patients of acromegaly in Spain is €9668. Even though surgery is the preferred option for treatment for a great number of patients, SAs must be added to the treatment regimen of the majority of such patients. The costs associated with this treatment are greater than the cost of treatment with SAs alone.

Validated assay for studying activity profiles of human liver UGTs after drug exposure: inhibition and induction studies.

UDP-glucuronsyltransferases (UGTs) are a family of conjugating enzymes that participate in the metabolism of many drugs. The study of potential drug-drug interactions involving UGTs has been largely hindered by the limited availability of selective functional assays for individual UGT enzymes. We propose a sensitive and reproducible procedure for the activity measurements of four major human hepatic UGT forms. The assays are based on analysis and quantification by high-performance liquid chromatography-tandem mass spectrometry of glucuronides formed from selective probe substrates, namely, beta-estradiol (UGT1A1, 3-glucuronide), 1-naphthol (UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). The analytical methods developed in the present study have been validated under good laboratory practice compliance following FDA recommendations. The assays can be easily applied to both phenotyping UGT reactions in liver-derived cellular and subcellular systems, and drug-drug interaction in vitro studies. Chemical inhibition of UGTs was tested in human liver microsomes at substrate concentrations lower than the corresponding K (M) values. Under these conditions, selective inhibition of UGT2B7 by fluconazole and low amitriptyline concentrations were observed, whereas diclofenac and quinidine were shown as non-enzyme-selective inhibitors of UGTs. Induction of UGTs was studied in primary human hepatocytes and HepG2 cells cultured in 96-well plates. Aryl hydrocarbon receptor ligands (except indirubin in hepatocytes) increased the UGT1A1 activity in both cell models. The highest effects were observed in HepG2 cells exposed to indirubin (21-fold over the control) and omeprazole or beta-naphthoflavone (about sixfold). Although variable effects were observed in other UGT enzymes, the degree of induction was generally lower than that for UGT1A1.

A new in vitro approach for the simultaneous determination of phase I and phase II enzymatic activities of human hepatocyte preparations.

Primary hepatocytes are still the best qualified in vitro system to anticipate drug metabolism in man. Recent advances in hepatocytes cryopreservation have notably increased their use not only for drug metabolism studies, but also for other applications such as cell transplantation. Evaluation of the drug-metabolizing competence of each hepatocytes preparation is needed. To date, the metabolic characterization of hepatocytes preparations relies on the assessment of phase I activities and the role of phase II enzymes receives little attention. A novel approach for the rapid assessment of the metabolic functionality of hepatocytes has been developed. A five-probe cocktail was used to simultaneously determine the enzymatic activities of major human phase I CYPs (CYP1A2, CYP2A6, CYP2C9, CYP2E1, CYP3A4), as well as two phase II enzymes: glucuronidase (UGTs) and sulfotransferase (SULT). Liquid chromatography/tandem mass spectrometry was used as the technique of choice for the determination of the enzymatic activities in a single run. Results showed that the method described herein permits a rapid assessment of the metabolic capabilities of human hepatocyte preparations as well as an estimation of the quality of freshly isolated or cryopreserved hepatocytes.

Functional assessment of the quality of human hepatocyte preparations for cell transplantation.

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepatocyte suspension was estimated by trypan blue staining. DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated dishes was quantified to estimate adherence capacity. Urea production was determined after incubating hepatocyte suspensions with 2 mM C1NH4 for 30 min. The cytochrome P450 function was assayed by a 30-min incubation of hepatocyte suspension with a cocktail mixture containing selective substrates for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4). The assay can be applied to both freshly isolated and cryopreserved hepatocyte suspensions, and the results are available within 1 h, which could help to make short-term decisions: 1) to assess the suitability for cell transplantation of a preparation of freshly isolated hepatocytes or a particular batch of thawed cells, or 2) to estimate the convenience of banking a particular cell preparation.

Monitoring pyrene excimers in lactose permease liposomes: revealing the presence of phosphatidylglycerol in proximity to an integral membrane protein.

In this study, we examined the annular lipid composition of the transmembrane protein lactose permease (LacY) from Escherichia coli. LacY was reconstituted into 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE) and 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-3-[Phospho-rac-(1-glycerol)] (POPG) and labeled with 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-Glycero-3-phosphoglycerol (PPDPG) at a 3:0.99:0.01 molar ratio. Pyrene excimer formation was monitored by exciting a single tryptophan mutant of the protein (T320W). The results suggest that POPG remains segregated in the vicinity of the protein, most likely forming part of the annular composition. The possible involvement of POPG in hydrogen binding with the protein, as well as the molecular mechanism of LacY, is also discussed in the context of the proteomic network theory.

Interfacial membrane effects of fluoroquinolones as revealed by a combination of fluorescence binding experiments and atomic force microscopy observations.

6-Fluoroquinolones are useful antimicrobial agents against gram-positive and gram-negative bacteria and some mycobacterial species as well. Although the diffusion through porins in gram-negative bacteria is well established, other mechanisms such as the hydrophobic pathway through the apolar regions of the bilayer and the self-promoted pathway appear to be relevant or concomitant with the hydrophilic pathway in many cases. This article discusses the interaction of ciprofloxacin (CPX) and two new synthesized compounds (M3CPX and M4CPX)-with a methyl group attached at the N3 and N4 positions of the piperazynil ring of the CPX-with liposomes and supported planar bilayers (SPBs) of Escherichia coli. Binding experiments using ANS revealed that the three compounds interact electrostatically with the bilayer. The variations in the electrostatic surface potential, which is always positive, were higher for M3CPX than for CPX or M4CPX. Related to that, the SPBs of E. coli were more affected by M3CPX than by the other two compounds, as judged by the analysis of the atomic force microcopy (AFM) images. The in situ injection of the three 6-fluoroquinolones (6-FQs) induced different changes in height, roughness (Ra), and area covered by the SPBs.

Surface planar bilayers of phospholipids used in protein membrane reconstitution: an atomic force microscopy study.

In this work, using atomic force microscopy (AFM), we have studied the influence of the temperature on the properties of the surface planar bilayers (SPBs) formed with: (i) the total lipid extract of Escherichia coli; (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC) (1:1, mol/mol); and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). According to the height profile analysis we performed, the height of the SPBs of DMPC:POPC were temperature dependent. Separated domains were observed in the SPBs of the POPE:POPG mixture and the E. coli lipid extract. The implication of those domains for the correct insertion of membrane proteins into proteoliposomes is discussed.

Preliminary atomic force microscopy study of two-dimensional crystals of lactose permease from Escherichia coli.

Lactose permease (LacY) of Escherichia coli is not only a paradigm for secondary transporters but also for difficulties in two-dimensional (2D) crystallization. In this work we present the progresses achieved in the observation of 2D crystals of wild-type LacY by atomic force microscopy (AFM). Crystals were obtained following reconstitution of LacY in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. Proteolipid sheets (PLSs) 6.4 nm in height were obtained after spreading the samples onto mica. Observations were carried out in liquid medium and in contact mode (CM-AFM). When the crystalline surfaces of the PLSs were imaged regular packing arrangements were observed. The back-Fourier transformation revealed the existence of various orientations mostly consistent with crystals possessing p2 symmetry and unit-cell dimensions: a=13.15 nm, b=16.74 nm, gamma=116 degrees. The characteristics, size, and shape of the repetitive motif could be compatible with dimers of this protein. These preliminary results are compared and discussed with previously reported 2D crystals observed by electron microscopy.

Effects of lactose permease of Escherichia coli on the anisotropy and electrostatic surface potential of liposomes.

The membrane transport protein lactose permease (LacY), a member of the Major Facilitator Superfamily (MFS) containing twelve membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes of: (i) 1,2-dimyristoyl-sn-glycero-3-phosphocoline (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in equimolar proportions; and (ii) Escherichia coli total lipid extract. The structural order of the lipid membranes, in the presence and absence of LacY, was investigated using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) evidenced: (i) the insertion of LacY into the bilayer; and (ii) a surface effect on the membranes. The most dramatic effects were observed when LacY was reconstituted in the E. coli lipid matrix. The effect of the protein on the electrostatic surface potential of each bilayer was also examined using a fluorescent pH indicator, 4-Heptadecyl-7-hydroxycoumarin (HHC). Changes in surface potential were enhanced in the presence of the substrate (i.e. lactose) only when the lipid matrices were charged. These results suggest a role for charged phospholipids (i.e. phosphatidylethanolamine or phosphatidylglycerol) in proton transfer to the amino acids involved in substrate translocation.