Drug Cost Avoidance Resulting from Participation in Clinical Trials: A 10-Year Retrospective Analysis of Cancer Patients with Solid Tumors.

The objective of this single-center retrospective study was to describe the clinical characteristics of adult patients with solid tumors enrolled in cancer clinical trials over a 10-year period (2010-2019) and to assess drug cost avoidance (DCA) associated with sponsors' contributions. The sponsors' contribution to pharmaceutical expenditure was calculated according to the actual price (for each year) of pharmaceutical specialties that the Vall d'Hebron University Hospital (HUVH) would have had to bear in the absence of sponsorship. A total of 2930 clinical trials were conducted with 10,488 participants. There were 140 trials in 2010 and 459 in 2019 (228% increase). Clinical trials of high complexity phase I and basket trials accounted for 34.3% of all trials. There has been a large variation in the pattern of clinical research over the study period, whereas, in 2010, targeted therapy accounted for 79.4% of expenditure and cytotoxic drugs for 20.6%; in 2019, immunotherapy accounted for 68.4%, targeted therapy for 24.4%, and cytotoxic drugs for only 7.1%. A total of four hundred twenty-one different antineoplastic agents were used, the variability of which increased from forty-seven agents in 2010, with only seven of them accounting for 92.8% of the overall pharmaceutical expenditure) to three hundred seventeen different antineoplastic agents in 2019, with thirty-three of them accounting for 90.6% of the overall expenditure. The overall expenditure on antineoplastic drugs in clinical care patients not included in clinical trials was EUR 120,396,096. The total cost of antineoplastic drugs supplied by sponsors in a clinical trial setting was EUR 107,306,084, with a potential DCA of EUR 92,662,609. Overall, clinical trials provide not only the best context for the progress of clinical research and healthcare but also create opportunities for reducing cancer care costs.

Use of Drugs in Clinical Practice and the Associated Cost of Cancer Treatment in Adult Patients with Solid Tumors: A 10-Year Retrospective Cohort Study.

BACKGROUND: Cancer is one of the leading causes of morbidity and mortality in the world. Its growing incidence and prevalence, as well as the advances in diagnostic and treatment tools, motivate an open debate about the economic burden it may place on health systems and have raised concerns about access to this technological innovation. There is a lack of information on the detailed costs of pharmacological treatment of cancer in our health setting. In this context, it is necessary to know the use of drugs in cancer treatment in conditions of real clinical practice. A real-word, evidence-based retrospective cohort study was conducted at Vall d'Hebron University Hospital (VHUH), the largest hospital complex in Catalonia, Spain, in order to determine the use of drugs and the associated cost in real clinical practice for the treatment of solid tumors in adult patients attended at this institution over 10 years (2010-2019). METHODS: This was a single-center retrospective cohort study of adult cancer patients attended in clinical practice at the Medical Oncology Department of VHUH between 1 January 2010 and 31 December 2019. Data of prescription, preparation, and cost of antineoplastic treatments were analyzed by pharmacological class (cytotoxic drugs, immunotherapy, targeted therapy, radiopharmaceuticals, and others), by antineoplastic agent, and by type of tumor. The number of patients and the pharmaceutical expenditure corresponding to all these subgroups were recorded. The cost per patient in each tumor location was also calculated. RESULTS: The study population included 13,209 patients with an overall pharmaceutical antineoplastic expenditure of EUR 120,396,097, increasing from 7.67% in relation to the total HUVH pharmaceutical expenditure in 2010 to 12.82% in 2019. By pharmacological class, the specific weight of the cost of targeted therapy is relevant (75.22% of pharmaceutical antineoplastic expenditure, 21.3% of patients) compared to the group of conventional cytotoxics (17.25% of pharmaceutical antineoplastic expenditure, 76.37% of patients), while immunotherapy has represented the largest relative increase, from 5% in 2014 to 12% in 2019. Eight targeted therapy drugs represented 50% of the costs of the targeted therapy drug class (palbociclib, trastuzumab, pertuzumab, bevacizumab, nivolumab, cetuximab, pembrolizumab, and trastuzumab emtansine). Eleven tumor sites accounted for 90% of the expenditure in 71% of all patients. Breast cancer had the highest expenditure during the study period (EUR 34,332,210) and at each individual year. Melanoma showed the highest increase, with 9.7% of total pharmaceutical antineoplastic expenditure in 2019 (2% of patients), representing a paradigm of the rising costs of cancer treatment due to the incorporation of new high-cost therapies. The average annual cost per patient was highly variable depending on the pathology. There was a growing increase in costs per patient in most tumor locations, particularly in patients with melanoma (from EUR 1922 in 2010 to EUR 37,020 in 2019), prostate cancer (from EUR 2992 in 2010 to EUR 14,118 in 2019), and non-small cell lung cancer (from EUR 3545 in 2010 to EUR 8371 in 2019). The relevance of the difference in monthly cost per patient that has been identified for the different intrinsic subtypes in breast cancer patients during 2019 (HER2+ EUR 2661/month, Luminal EUR 881/month, Triple negative EUR 386/month) makes us consider suggesting differentiated reimbursement rates for certain clinical conditions. Finally, support treatment with antiemetic drugs, erythropoietin stimulating agents, granulocyte-colony stimulating factor (G-CSF), and bone resorption inhibitors has involved a cost of EUR 5,751,910, which represents 4.6% of the overall pharmacological cost of cancer treatment. CONCLUSION: This study provides detailed insights on the oncological pharmaceutical expenditure for the treatment for solid tumors in the VHUH, based on real cost information from our hospital practice and for all antineoplastic therapies and types of solid tumors. This type of information on all the different types of cancer can be useful to better understand the economic burden of the disease and can be decisive for allocating public resources and funds for research, especially in those areas where information is scarce and therefore where further studies are needed. The contribution to knowledge of the cost of oncology therapy is of great value due to its realism and scope.

Use of Phycobiliproteins from Atacama Cyanobacteria as Food Colorants in a Dairy Beverage Prototype.

The interest of the food industry in replacing artificial dyes with natural pigments has grown recently. Cyanobacterial phycobiliproteins (PBPs), phycoerythrin (PE) and phycocyanin (PC), are colored water-soluble proteins that are used as natural pigments. Additionally, red PE and blue PC have antioxidant capabilities. We have formulated a new food prototype based on PBP-fortified skim milk. PBPs from Andean cyanobacteria were purified by ammonium sulfate precipitation, ion-exchange chromatography, and freeze-drying. The stability of PE and PC was evaluated by changes in their absorption spectra at various pH (1-14) and temperature (0-80 °C) values. Purified PBPs showed chemical stability under pH values of 5 to 8 and at temperatures between 0 and 50 °C. The antioxidant property of PBP was confirmed by ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical ion scavenging, and FRAP (Ferric Antioxidant Power) assays. The absence of PBP toxicity against Caenorhabditis elegans was confirmed up to 1 mg PBP/mL. Skim milk fortified with PE obtained a higher score after sensory tests. Thus, a functional food based on skim milk-containing cyanobacterial PBPs can be considered an innovative beverage for the food industry. PBPs were stable at an ultra-high temperature (138 °C and 4 s). PBP stability improvements by changes at its primary structure and the incorporation of freeze-dried PBPs into sachets should be considered as alternatives for their future commercialization.

Palbociclib and ribociclib in breast cancer: consensus workshop on the management of concomitant medication.

Drug-drug interactions are of significant concern in clinical practice in oncology, particularly in patients receiving Cyclin-dependent kinase (CDK) 4/6 inhibitors, which are typically exposed to long-term regimens. This article presents the highlights from the 'First Workshop on Pharmacology and Management of CDK4/6 Inhibitors: Consensus about Concomitant Medications'. The article is structured into two modules. The educational module includes background information regarding drug metabolism, corrected QT (QTc) interval abnormalities, management of psychotropic drugs and a comprehensive review of selected adverse effects of palbociclib and ribociclib. The collaborative module presents the conclusions of the five working groups, each of which comprised five experts from different fields. From these conclusions positive lists of drugs for treating common comorbid conditions that can be safely administered concomitantly with palbociclib and/or ribociclib were developed.

Clinical, Biochemical, and Genetic Characteristics of "Nonclassic" Apparent Mineralocorticoid Excess Syndrome.

Context: Classical apparent mineralocorticoid excess (AME) is a rare recessive disorder, caused by severe 11ß-hydroxysteroid dehydrogenase type 2 enzyme (11ß-HSD2) deficiency. AME manifests as low-renin pediatric hypertension, hypokalemia and high cortisol/cortisone (F/E) ratio. Objective: To evaluate nonclassic AME (NC-AME) due to partial 11ß-HSD2 insufficiency and its association with hypertension, mineralocorticoid receptor (MR) activation, and inflammatory parameters. Design: Cross-sectional study. Setting: Primary care cohort. Participants: We recruited 127 adolescents and adults. Subjects with secondary hypertension were excluded. We measured clinical, biochemical, renal, vascular, and inflammatory variables. Sequencing of HSD11B2 gene was performed in all subjects. Main Outcome Measure: NC-AME. Results: Serum F/E ratio was positively associated with systolic blood pressure (BP), microalbuminuria, and high-sensitivity C-reactive protein (hs-CRP). Serum cortisone correlated with MR activation parameters even when adjusted for age, body mass index, and sex: lower cortisone with higher potassium excretion (partial r = -0.29, P = 0.002) and with lower plasma renin activity (PRA) (partial r = 0.29, P = 0.001). Consistently, we identified 9 in 127 subjects (7.1%) with high F/E ratios (first quartile) and low cortisone (last quartile), suggestive of NC-AME. These subjects had higher systolic BP, 141.4 ± 25.7 mm Hg vs 127.3 ± 18.1 mm Hg, P = 0.03; lower PRA, 0.36 ± 0.19 ng/L*s vs 0.64 ± 0.47 ng/L*s, P < 0.0001; and greater potassium excretion, microalbuminuria, hs-CRP, and plasminogen activator inhibitor. We only found in 2 out of 9 subjects with NC-AME heterozygous mutations in the HSD11B2 gene. Conclusions: These findings suggest a spectrum of partial 11ß-HSD2 insufficiency in a primary care cohort without the classic phenotype and genotype of AME. NC-AME may represent a phenotype of MR activation and cardiovascular risk, suggesting that these subjects could be treated with MR antagonists.

Sodium Intake Is associated With Endothelial Damage Biomarkers and Metabolic Dysregulation.

BACKGROUND: Mounting evidence has associated high sodium (HS) intake with hypertension, cardiovascular disease, and stroke. We investigated whether HS intake modulates the parameters of endothelial damage, inflammation, and oxidative stress. METHODS: We used a cross-sectional study design including 223 Chilean subjects (6.9-65.0 years old). We measured aldosterone, renin activity, cortisol, cortisone, adiponectin, leptin, hsCRP, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor type 1 (PAI-1), metalloproteinase (MMP)-9 and MMP-2 activity, and malondialdehyde. Sodium and creatinine were measured in 24-hour urine samples. The subjects were divided by sodium intake, high sodium (HS): ≥150 mEq/day, n = 118, and adequate sodium (AS): <150 mEq/day, n = 105. RESULTS: We observed a positive correlation between urinary sodium excretion and blood pressure (r = 0.1669, P = 0.0124 for systolic and r = 0.2416, P = 0.0003 for diastolic), glycemia (r = 0.2660, P < 0.0001), and triglycerides (r = 0.1604, P = 0.0175) and a highly significant correlation between sodium excretion and PAI-1 (r = 0.2701, P < 0.0001). An inverse correlation was observed between urinary sodium and HDL-cholesterol (r = -0.2093, P = 0.0018) and adiponectin (r = -0.2679, P < 0.0001). In a linear regression model, urinary sodium excretion remained significantly associated with PAI-1 values even after adjusting for age, gender, and BMI. The HS group had higher blood pressure, glycemia, HOMA-IR, atherogenic index of plasma, and PAI-1 values than the group with AS intake. CONCLUSIONS: HS intake is associated with endothelial damage (high PAI-1) and metabolic dysregulation. On the other hand, inflammation and oxidative stress parameters are not modified by sodium intake.

Serum Cortisol and Cortisone as Potential Biomarkers of Partial 11ß-Hydroxysteroid Dehydrogenase Type 2 Deficiency.

BACKGROUND: Pathogenic variations in HSD11B2 gene triggers the apparent mineralocorticoid excess syndrome (AME). There is scarce information regarding the phenotypes of subjects carrying heterozygous pathogenic variants in HSD11B2 gene. We investigated if serum cortisol/cortisone (F/E) ratio and cortisone are useful for identifying partial 11ßHSD2 deficiency in those heterozygous subjects. METHODS: We studied two patients diagnosed with AME and their families carrying either D223N or R213C mutation. We also evaluated 32 healthy control subjects (13 children and 19 adults) to obtain normal references ranges for all measured variables. Case 1: A boy carrying D223N mutation in HSD11B2 gene and Case 2: A girl carrying R213C mutation. We assessed serum F/E ratio and cortisone by HPLC-MS/MS, aldosterone, plasma-renin-activity(PRA), electrolytes, and HSD11B2 genetic analyses. RESULTS: The normal values (median [interquartile range]) in children for serum F/E and cortisone (µg/dl) were 2.56 [2.21-3.69] and 2.54 [2.35-2.88], and in adults were 4.42 [3.70-4.90] and 2.23 [1.92-2.57], respectively. Case 1 showed a very high serum F/E 28.8 and low cortisone 0.46 µg/dl. His mother and sister were normotensives and heterozygous for D223N mutation with high F/E (13.2 and 6.0, respectively) and low cortisone (2.0 and 2.2, respectively). Case 2 showed a very high serum F/E 175 and suppressed cortisone 0.11 µg/dl. Her parents and sister were heterozygous for the R213C mutation with normal phenotype, but high F/E and low cortisone. Heterozygous subjects showed normal aldosterone, PRA, but lower fractional excretion of sodium and urinary Na/K ratio than controls. CONCLUSION: Serum F/E ratio and cortisone allow to identify partial 11ßHSD2 deficiencies, as occurs in heterozygous subjects, who would be susceptible to develop arterial hypertension.

Cortisol/cortisone ratio and matrix metalloproteinase-9 activity are associated with pediatric primary hypertension.

OBJECTIVE: To identify novel biomarkers associated with pediatric primary hypertension. METHODS: We recruited 350 participants (4-16 years). Anthropometric parameters and aldosterone, plasma renin activity, cortisol, cortisone, Homeostasis Model Assessment Insulin Resistance (HOMA-IR), high-sensitivity C-reactive protein, adiponectin, IL-6, plasminogen activator inhibitor type 1 levels and matrix metalloproteinase-9 and matrix metalloproteinase-2 (MMP-9 and MMP-2) activities were measured. Genomic DNA was isolated. Patients with altered glucose metabolism, severe obesity [BMI-SD score (BMI-SDS) > 2.5], renovascular disease, primary aldosteronism and apparent mineralocorticoid excess syndrome were excluded. RESULTS: In selected participants (n = 320), SBP was positively correlated with BMI-SDS (r = 0.382, P < 0.001), HOMA-IR (r = 0.211, P < 0.001), MMP-9 activity (r = 0.215, P < 0.001) and the cortisol/cortisone ratio (r = 0.231, P < 0.001). DBP showed similar correlations with these variables. No correlation was observed with aldosterone or plasma renin activity. Participants were categorized as hypertensive (n = 59) or nonhypertensive (n = 261). In the univariate analysis, hypertensive patients had higher BMI-SDS (P < 0.001), HOMA-IR (P < 0.001), high-sensitivity C-reactive protein (P < 0.001), MMP-9 activity (P < 0.001), plasminogen activator inhibitor type 1 (P < 0.001) and cortisol/cortisone ratio (P < 0.001) than nonhypertensive patients. Multiple regression analysis showed that the variables that remained associated with hypertension were higher BMI-SDS [odds ratio (OR) = 3.74; 95% confidence interval (CI) = 1.84-7.58], a higher cortisol/cortisone ratio (OR = 3.92; 95% CI = 1.98-7.71) and increased MMP-9 activity (OR = 4.23; 95% CI = 2.15-8.32). CONCLUSION: We report that MMP-9 activity and the cortisol/cortisone ratio were higher in pediatric primary hypertensive patients, and these associations were independent of the effect of obesity. The potential role of these novel biomarkers in predicting hypertension risk and blood pressure regulation warrants further investigation.

Usefulness and Pitfalls in Sodium Intake Estimation: Comparison of Dietary Assessment and Urinary Excretion in Chilean Children and Adults.

BACKGROUND: High sodium intake has been associated with various noncommunicable disease like hypertension, cardiovascular disease, or stroke. To estimate accurately sodium intake is challenging in clinical practice. We investigate the usefulness and limitations of assessing sodium intake simultaneously by dietary assessment and urinary samples in both children and adults. METHODS: We used a cross-sectional study design inviting 298 Chilean subjects (74 children and 222 adults) aged between 9 and 66 years of both genders. Sodium intake by dietary assessment was obtained from Chilean food composition data, based on FAO tables. Sodium and creatinine excretion were measured in 24-hour urine samples, in all participants. RESULTS: Adequate urinary collection was obtained in 81% of children (59/74) and 61% of adults (135/222). The mean sodium intake by dietary assessment was similar to the sodium excretion in 24 hours (3,121±1,153mg/d vs. 3,114±1,353mg/24h, P = nonsignificant) in children but was significantly lower (3,208±1,284mg/d vs. 4,160±1,651mg/24h, P < 0.001) in adults. In both children and adults, sodium intake correlated with urinary sodium excretion (r = 0.456, P < 0.003 and r = 0.390, P < 0.001, respectively). Secondary analyses also suggested that the dietary assessment was more inaccurate in overweight adult subjects. CONCLUSIONS: Our results showed that average sodium intake was higher than recommended in both children and adults (WHO ≤2,000mg/d). The sodium intake estimated by dietary assessment correlated with urinary excretion in all subjects, but in obese adults was more inaccurate than in children. Future studies to validate the appropriate test to assess sodium intake by age and nutritional status are warranted.

Citosine-Adenine-Repeat Microsatellite of 11ß-hydroxysteroid dehydrogenase 2 Gene in Hypertensive Children.

BACKGROUND: The impairment of 11ß-hydroxysteroid dehydrogenase type 2 enzyme (11ßHSD2) results in an inefficient conversion of cortisol to cortisone, which triggers hypertension. Cytosine-adenine repeat (CA repeat) microsatellite has been associated with low HSD11B2 gene expression. AIM: To determine whether the CA-repeat length in intron 1 affect the serum cortisol to cortisone (F/E) ratio and/or blood pressure (BP) levels in pediatric subjects. SUBJECTS AND METHODS: Eighty-one hypertensive (HT) and 117 normotensive (NT) subjects participated in this study. We measured BP levels, as well as the F and E and F/E ratio in morning sera and 12-hour urine samples. The length of CA repeats was determined through fragment analysis. We compared the allele distribution between the HT and NT groups, and the patients were dichotomized into groups with short alleles (S) (<21 CA repeats) or long alleles (L), and also in groups according genotype (allele combination: S/S and S/L + L/L). RESULTS: We found no differences in the distribution of CA-repeat allelic length between the NT and HT groups (P = 0.7807), and there was no correlation between the CA-repeat allelic length and BP (P = 0.1151) levels or the serum F/E ratio (P = 0.6778). However, the serum F/E ratio was higher in the HT group than in the NT group (P = 0.0251). The serum F/E ratio was associated with systolic BP index independent of body mass index only in HT group. CONCLUSIONS: The CA-repeat length did not influence BP levels or serum F/E ratios in pediatric subjects. However, the serum F/E ratio was associated with BP, suggesting a role of 11ßHSD2 in mineralocorticoid hypertension.

The Expression of RAC1 and Mineralocorticoid Pathway-Dependent Genes are Associated With Different Responses to Salt Intake.

BACKGROUND: Rac1 upregulation has been implicated in salt-sensitive hypertension as a modulator of mineralocorticoid receptor (MR) activity. Rac1 could affect the expression of oxidative stress markers, such as hemoxigenase-1 (HO-1) or nuclear factor-B (NF-κB), and the expression of neutrophil gelatinase-associated lipocalin (NGAL), a cytokine upregulated upon MR activation. AIM: We evaluated RAC1 expression in relation of high salt intake and association with MR, NGAL, HO-1, and NF-κB expression, mineralo- and glucocorticoids levels, and inflammatory parameters. SUBJECTS AND METHODS: We studied 147 adult subjects. A food survey identified the dietary sodium (Na) intake. RAC1 expression was considered high or low according to the value found in normotensive subjects with low salt intake. We determined the gene expression of RAC1, MR, NGAL, HO-1, NF-κB, and 18S, isolated from peripheral leukocytes. We measured aldosterone, cortisol, sodium, potassium excretion, metalloproteinase (MMP9 y MMP2), and C-reactive protein. RESULTS: We identified 126 subjects with high Na-intake, 18 subjects had high, and 108 low-RAC1 expression. The subjects with high-RAC1 expression showed a significant increase in MR (P = 0.0002), NGAL (P < 0.0001) HO-1 (P = 0.0004), and NF-κB (P < 0.0001) gene expression. We demonstrated an association between RAC1 expression and MR (R sp 0.64; P < 0.0001), NGAL (R sp 0.48; P < 0.0001), HO-1 (R sp 0.53; P < 0.0001), and NF-κB (R sp0.52; P < 0.0001). We did not identify any association between RAC1 and clinical or biochemical variables. CONCLUSIONS: RAC1 expression was associated with an increase in MR, NGAL, NF-κB, and HO-1 expression, suggesting that RAC1 could be a mediator of cardiovascular damage induced by sodium, and may also useful to identify subjects with different responses to salt intake.

LC-MS/MS Method for the Simultaneous Determination of Free Urinary Steroids.

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) converts cortisol to cortisone. In contrast, 5α and 5ß reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC-MS/MS being the reference method. The 11ß-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11ß-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1-120 ng/mL for cortisol and cortisone, and 1-120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85-105 %, respectively. Our LC-MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11ß-HSD2 and 11ß-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.

Polymorphisms in the RAC1 gene are associated with hypertension risk factors in a Chilean pediatric population.

BACKGROUND: The GTPase Rac1 has been implicated in hypertension as a modulator of mineralocorticoid receptor activity. Our aim is to investigate the frequency of polymorphisms rs10951982 (intron 1, G>A) and rs836478 (intron 3, T>C) in the RAC1 gene and perform association studies with clinical and biochemical parameters in a Chilean pediatric cohort. METHODS: Two hundred two normotensive (NT) subjects (aged 4-16 years) were divided into 2 groups: NT subjects with hypertensive parents (NH; n = 103) and NT subjects with NT parents (NN; n = 99). We measured markers of inflammation (high-sensitivity C-reactive protein, interleukin 6 (IL-6), interleukin 8, and tumor necrosis factor α), endothelial damage (Plasminogen activator inhibitor-1 metalloproteinase-9, and metalloproteinase-2), and oxidative stress (malondialdehyde). Data were expressed as median and interquartile range (IQR). RESULTS: We found differences in polymorphism rs836478 (intron 3, C>T) in both genotypic (χ(2) = 15.2, 2 df; P = 0.0005) and allelic (X(2)=5.5, 1 df; P = 0.01) frequencies in NH vs. NN subjects. NH subjects with a TT genotype showed increase MMP9 expression (median = 2.3, IQR - 1.6-3.2; vs. median = 1.6, IQR = 1.6-2.3 AU; P = 0.01) and lower IL-6 expression (median = 8.8, IQR = 7.0-11.8; vs. median = 12.1, IQR = 8.2-14.7 pg/ml; P = 0.02) compared with subjects with TC/CC genotype. No difference in the allelic frequency distribution was seen in the polymorphism rs10951982 (NH vs. NN: χ(2)=0.2, 1 df; P = 0.6). For this SNP, NN subjects with GA/AA genotype showed decreased diastolic BP indexes compared with subjects with native GG genotype (median = 1.08, IQR = 1.0-1.2; vs. median = 0.99, IQR = 0.94-1.1; P = 0.02). CONCLUSIONS: We report the frequency of polymorphisms rs836478 and rs10951982 of the RAC1 gene in a Spanish-Amerindian cohort. The polymorphism rs836478 was associated with an increased expression in markers of inflammation and endothelial damage (MMP9 and IL-6) in pediatric subjects with a hypertensive genetic background.

Identification of novel 11ß-HSD1 inhibitors by combined ligand- and structure-based virtual screening.

11 beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts cortisone to cortisol in a NADPH dependent manner. Overexpression of 11ß-HSD1 in key metabolic tissues is related to the development of type 2 diabetes, obesity, hypertension and metabolic syndrome. Using crystal structures of human 11ß-HSD1 in complex with inhibitors as source of structural information, a combined ligand and structure-based virtual screening approach was implemented to identify novel 11ß-HSD1 inhibitors. A selected group of compounds was identified in silico and further evaluated in cell-based assays for cytotoxicity and 11ß-HSD1 mediated cortisol production inhibitory capacity. The expression of 11ß-HSD1 and 11ß-HSD2 in human LS14 adipocytes was assessed during differentiation. Biological evaluation of 39 compounds in adipocytes and steroids quantification by HPLC-MS/MS identify 4 compounds that exhibit 11ß-HSD1 mediated cortisol production inhibitory activity with potencies in the micromolar range. Two compounds showed to be selective for the 11ß-HSD1 reductase activity and over 11ß-HSD2 isoform, and thus represent novel leads for the development of more active derivatives with higher efficacies targeting intracellular cortisol levels in type 2 diabetes and metabolic syndrome.

Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro.

BACKGROUND: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes. METHODS: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids. RESULTS: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients. CONCLUSIONS: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.

Distribution of pesticides and PCBs in sediments of agricultural drains in the Culiacan Valley, Sinaloa, Mexico.

Agriculture is one of the most important economic activities in Sinaloa, Mexico. The Culiacan Valley is an extensive agricultural region characterized by a variety of crops with high-yield productions. In this study, concentrations of organochlorine (OCPs) and organophosphorus (OPs) pesticides and polychlorobiphenyls (PCBs) were determined in sediments of the agricultural drainage system of Culiacan Valley. Overall, 32 compounds were detected, with concentrations widely ranging from 0.03 to 1 294 ng g(-1) dry weight. OCP concentrations (15) ranged from 0.1 to 20.19 ng g(-1) dw. OP concentrations (8) ranged from 0.03 to 1294 ng g(-1) dw, and diazinon was the compound with the highest concentration. PCB concentrations were also determined and varied from 0.05 to 3.29 ng g(-1) dw. Other compounds detected included permethrin, triadimefon, and fipronil. The central zone registered the higher concentrations and the greatest number of compounds, which could be related to the occurrence of horticultural fields in this zone. According to sediment quality guidelines, the compounds exceeding the probable effect level were γ-HCH, p,p'-DDT and p,p'-DDE, while the pesticides above the maximum permissible concentration were endosulfan, azinphos methyl, diazinon, dichlorvos, and permethrin. Although Sinaloa is an important agricultural crop producer in northwest Mexico, there are not many studies dealing with pesticide distribution in agricultural areas.

11ß-hydroxysteroid dehydrogenase type 2 polymorphisms and activity in a Chilean essential hypertensive and normotensive cohort.

BACKGROUND: 11ß-hydroxysteroid dehydrogenase type 2 enzyme (11ß-HSD2) inactivates cortisol (F) to cortisone (E); its impairment is associated with hypertension. We reported that 15.7% of the Chilean essential hypertensives possessed a high F/E ratio suggesting a partial deficit in 11ß-HSD2 activity. It has been reported that the G534A(Glu178/Glu) polymorphism in the HSD11B2 gene is associated with hypertension. Investigate the frequency of the G534A polymorphism and its correlation with the glucocorticoid profile in Chilean essential hypertensive and normotensive subjects. METHODS: Essential hypertensive outpatients (n = 232) and normotensive subjects (n = 74) were recruited. A change in the AluI restriction enzyme digest pattern, caused by the presence of the G534A polymorphism, was utilized to screen DNA isolated from leukocytes within the cohort before confirmation by sequencing. Plasma renin activity (PRA), serum aldosterone, F, and E were measured by radioimmunoassay. Urinary tetrahydrocortisol (THF), 5α-tetrahydrocortisol (5α-THF), and tetrahydrocortisone (THE) were measured by gas chromatography-mass spectrometry. RESULTS: G534A polymorphism frequency was similar between hypertensive patients (19 of 232; 8.2%) and normotensive subjects (7 of 74; 9.5%). When categorized by presence or absence of the G534A polymorphism, no significant differences in the serum F/E ratio or other measured biochemical variables were detected. Despite a previous report that the G534A polymorphism is associated with a neighboring C468A (Thr156/Thr) polymorphism, analysis within our cohort showed that only one patient in each group presented with this double polymorphism. CONCLUSIONS: We report the frequency of the G534A polymorphism in the Spanish-Amerindian population. No correlation was detected between this polymorphism and the presence of hypertension and biochemical parameters in this Chilean cohort.

A new presentation of the chimeric CYP11B1/CYP11B2 gene with low prevalence of primary aldosteronism and atypical gene segregation pattern.

Familial hyperaldosteronism type I is caused by an unequal crossover of 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, giving rise to a chimeric CYP11B1/CYP11B2 gene (CG). We describe a family carrying a CG with high levels of free 18-hydroxycortisol but low prevalence of primary aldosteronism (PA) and an atypical CG inheritance pattern in a family of 4 generations with 16 adults and 13 children, we measured the arterial blood pressure, serum aldosterone, and plasma renin activity and then calculated the serum aldosterone:plasma renin activity ratio and urinary free 18-hydroxycortisol. We identified the CG by long-extension PCR and predicted its inheritance pattern. The CG was found in 24 of 29 subjects (10 children and 14 adults). In CG+ patients, hypertension and high 18-hydroxycortisol were prevalent (83% and 100%, respectively). High serum aldosterone:plasma renin activity ratio was more frequent in pediatric than adult patients (80% versus 36%; P<0.001). An inverse association between serum aldosterone:plasma renin activity ratio and age was observed (r=-0.48; P=0.018). Sequence analysis identified the CYP11B1/CYP11B2 crossover in a 50-bp region spanning intron 3 of CYP11B1 and exon 4 of CYP11B2. The CG segregation differs from an autosomal disease, showing 100% of CG penetrance in generations II and III. Statistical analysis suggests that inheritance pattern was not attributed to random segregation (P<0.001). In conclusion, we describe a family with an atypical CYP11B1/CYP11B2 gene inheritance pattern and variable phenotypic expression, where the majority of pediatric patients have primary aldosteronism. Most adults have normal aldosterone and renin levels, which could mask them as essential hypertensives.