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Total pancreatectomy with islet autotransplantation is a complex surgical approach for acute recurrent or chronic pancreatitis that frequently triggers extreme thrombocytosis (platelets ≥ 1000 × 109 /L). Thrombocytosis can be prothrombotic, so cytoreductive hydroxyurea is often initiated after this surgery; however, optimal dosing strategy and efficacy are unknown. This prospective pilot study characterized the pharmacokinetics of hydroxyurea after this procedure in children. It also compared them with previously published pediatric parameters in sickle cell anemia (SCA), the disease in which pediatric hydroxyurea pharmacokinetics have primarily been studied. Plasma hydroxyurea levels were quantified in 14 participants aged 4-19 years using high-performance liquid chromatography. Blood collections were scheduled 20 minutes, 1 hour, and 4 hours after the first dose, on pharmacokinetic day 1 (PK1), and again 2-3 months later if still on hydroxyurea (PK2). Six participants had PK1 and PK2 data at all 3 postdose timed collections, 5 only had PK1 samples, and 3 only had PK2 samples. Total pancreatectomy with islet autotransplantation participants had reduced and delayed absorption compared with sickle cell anemia participant data from the Hydroxyurea Study of Long-Term Effects, regardless of timing or dosing methodology. Total pancreatectomy with islet autotransplantation participants had different pharmacokinetic profiles at PK1 versus PK2, with lower dose-normalized exposures than previously reported in sickle cell anemia. These results suggest variability exists in hydroxyurea absorption and bioavailability in total pancreatectomy with islet autotransplantation patients, suspected to be primarily because of Roux-en-Y reconstruction, and suggest that more pharmacokinetic data are needed for scenarios when hydroxyurea is prescribed to children without sickle cell anemia.

Introduction: Hydroxyurea is a potent therapeutic agent for sickle cell anemia (SCA), and treatment at maximum tolerated dose (MTD) is becoming the standard of care. Hydroxyurea exerts its disease-modifying effects primarily through induction of fetal hemoglobin (HbF), although the cellular and molecular mechanisms by which hydroxyurea increases HbF expression remain unclear. Children with SCA treated with hydroxyurea at MTD have substantial phenotypic variation, however, as some have higher HbF responses than others. We hypothesized that unknown quantitative trait loci modulate the pharmacological induction of HbF, so we performed a large genome wide association study (GWAS) of hydroxyurea-associated HbF responses for children with SCA treated prospectively with dose escalation to MTD. Methods: We analyzed genomic DNA from 831 children with SCA enrolled in pediatric research trials from the US (HUSTLE, SWiTCH, TWiTCH), the Caribbean (EXTEND, SACRED) and sub-Saharan Africa (REACH, NOHARM); all of these trials reported robust treatment responses with average HbF >20%. Study participants received hydroxyurea with dose escalation to MTD based on mild myelosuppression. Whole blood DNA was genotyped using the H3Africa SNP array (Illumina) with whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform. A transformed z-score for each study cohort gave a standardized measure of HbF induction relative to their steady-state level and their treatment HbF level at MTD. These standardized z-score HbF values were then used as a continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, using age, hydroxyurea dose at MTD, and sex as co-variates. We first performed an initial GWAS discovery using hydroxyurea response data from four distinct African populations (n=377). Single nucleotide variants (SNVs) with nominal significance (p Results: In the discovery GWAS step, no variant passed genome wide significance (p Conclusions: This large GWAS using global cohorts of children with SCA and robust prospective HbF phenotype data has identified genetic predictors of HbF hydroxyurea treatment responses. Three novel genetic loci, PTPRD, RPH3AL, and ELL2 have SNVs associated with lower HbF responses. PTPRD is a protein tyrosine phosphatase receptor involved in cellular processes such as cell growth and differentiation, while RPH3AL, a rabphilin 3A like protein, is known to be involved in calcium-ion-dependent exocytosis. ELL2 is an elongation factor for RNA polymerase II and could modify RNA processing under the cytostatic effects of hydroxyurea. These genes and variants will be investigated to determine how they impact individual HbF responses to hydroxyurea treatment. Disclosures Aygun: National Heart, Lung, and Blood Institute: Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Research Funding; National Institute of Nursing Research: Research Funding; Patient-Centered Outsomes Research Institute: Research Funding.

Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Square root transformed HbF values were the continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, with both age and sex as co-variates. The GWAS approach included 3 distinct steps. First, we performed two independent GWAS discovery steps using distinct African populations; these were designated Discovery I (N=211) and Discovery II (223). Second, only SNVs that were significant (p Results: From the combined SNP and WES dataset, 8 BCL11A variants passed genome wide significance (p Conclusions: Our large GWAS of HbF with diverse global cohorts of children with SCA from Africa, the United States, and the Caribbean validated the strong associations of HbF with common genetic variants near the BCL11A and HBS1L-MYB gene loci. We also identified two novel gene loci, ITGA1 and RUNX1T1, that have statistical associations with HbF expression. The RUNX1T1 gene is a broad transcriptional corepressor known to impact myeloid differentiation in hematopoiesis, while ITGA1 encodes the integrin alpha subunit of a cell-surface receptor involved in cell-cell adhesion and inflammation. Both of these genes represent novel loci that may be involved in the regulation of HbF expression in children with SCA and should be investigated further using cellular and animal models. Disclosures No relevant conflicts of interest to declare.

Hydroxyurea is FDA-approved and now increasingly used for children with sickle cell anemia (SCA), but dosing strategies, pharmacokinetic (PK) profiles, and treatment responses for individual patients are highly variable. Typical weight-based dosing with step-wise escalation to maximum tolerated dose (MTD) leads to predictable laboratory and clinical benefits, but often takes 6 to 12 months to achieve. The Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154) was a single-center study designed to prospectively validate a novel personalized PK-guided hydroxyurea dosing strategy with a primary endpoint of time to MTD. Enrolled participants received a single oral 20 mg/kg dose of hydroxyurea, followed by a sparse PK sampling approach with three samples collected over three hours. Analysis of individual PK data into a population PK model generated a starting dose that targets the MTD. The TREAT cohort (n = 50) was young, starting hydroxyurea at a median age of 11 months (IQR 9-26 months), and PK-guided starting doses were high (27.7 ± 4.9 mg/kg/d). Time to MTD was 4.8 months (IQR 3.3-9.3), significantly shorter than comparison studies (p

Background Total pancreatectomy with islet autotransplantation (TPIAT) is a surgical treatment for chronic recurrent pancreatitis that involves splenectomy, pancreatectomy and creation of a Roux-en-Y, followed by re-injection of the pancreatic islets into the portal vein. Postoperatively, patients develop a sustained and extreme thrombocytosis (ExT) with platelets ≥1000 K/μL, a response more exaggerated than the typical post-splenectomy course (Gurria JP et al, Pancreas 2019). Empiric aspirin (antiplatelet effect) and hydroxyurea (cytoreduction) are initiated postoperatively per a standard protocol. The pharmacokinetic (PK) and pharmacodynamic (PD) profiles of hydroxyurea have not been studied in children other than young children with sickle cell disease; neither dose nor dose interval have been evaluated in TPIAT patients. Recently a population PK model was developed to support individualized hydroxyurea dosing in patients with sickle cell anemia (SCA; Dong M et al, Br J Clin Pharmacol 2016). In a prospective evaluation as part of the Therapeutic Response Evaluation and Adherence Trial (TREAT, McGann PT et al, Am J Hematol 2019), PK-guided individualized dosing resulted in better clinical and laboratory benefits than with conventional weight-based dosing. This study aimed to determine if a hydroxyurea PK model could be developed for non-SCA children dosed postoperatively for control of TPIAT-associated ExT, and if so, to compare it to previously published models for SCA. Methods A prospective single-site pilot study was performed in patients ages 0-21 years who underwent TPIAT between April 2018 and June 2019. Whole blood was collected via finger stick or venipuncture at 3 time points (20 minutes, 1 hour, 4 hours) after the initial hydroxyurea dose (15-20 mg/kg), given between postoperative day 5-7 (PK1). Plasma hydroxyurea was quantified by high performance liquid chromatography on 150-200 μL plasma. Testing was repeated once 2-6 months postoperatively (PK2) to determine whether PK profiles changed over time in relation to surgery. PK analyses and estimation of the area under the concentration-time curve (AUC) as a measure of exposure were performed using MW/Pharm (Mediware, Prague, Czech Republic). PK data from HUSTLE (NCT00305175) were used for comparison to SCA patients. Results Of 19 enrolled subjects, 15 had evaluable results: 7 had both PK collections while 8 had a single collection (5 only had PK1, 3 only had PK2). All 5 patients with only PK1 measurements had discontinued hydroxyurea by the time PK2 samples would have been collected, whereas those with only PK2 all had unsuccessful PK1 collection attempts. Mean age was 13.5 years [standard deviation (SD) 5] with mean weight-based dose 14 mg/kg (SD 3.4). Serum creatinine was normal for age in all patients. Hydroxyurea suspension was delivered via jejunal tube at PK1 while oral tablets were used at PK2 per surgical protocol. The AUC was 60.1 h*μg/ml at PK1 and 68.6 h*μg/ml at PK2. When compared to HUSTLE cohort (Figure 1), TPIAT subjects overall had lower drug exposure (AUC), worse for PK1 than PK2, especially when hydroxyurea concentration was normalized to dose. TPIAT patients also appear to have had slower absorption, evidenced by still-rising hydroxyurea concentrations after 1 hour, compared to 20-30 minute peak concentrations in HUSTLE. Conclusions TPIAT subjects treated empirically with hydroxyurea to modulate ExT have decreased absorption and overall lower concentrations and drug exposure relative to our SCA population. Lower bioavailability and/or altered absorption may in part be caused by differences in formulation (suspension versus tablet) and route of administration (oral versus jejunal) along with postoperative anatomic changes not present in the SCA cohort, since Roux-en-Y may alter drug absorption and metabolism (Rogers CC et al, Clin Transpl2008). It is unknown whether higher enteral hydroxyurea doses would increase exposure and effect. Additional evaluation of the absorption characteristics and PK/PD of hydroxyurea in TPIAT patients, as well as efficacy, are urgently needed. Figure 1 Disclosures Ware: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Nova Laboratories: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: DSMB; Bristol Myers Squibb: Other: Research Drug Donation; Addmedica: Other: Research Drug Donation. OffLabel Disclosure: Hydroxyurea to reduce extreme thrombocytosis after splenectomy/pancreatic islet cell transplant in children

Hydroxyurea is a potent disease-modifying therapeutic agent with efficacy for the treatment of sickle cell anemia. When administered at once-daily oral doses that lead to mild marrow suppression, hydroxyurea leads to substantial and sustained fetal hemoglobin induction, which effectively inhibits erythrocyte sickling. When escalated to maximum tolerated dose, hydroxyurea has proven laboratory and clinical effects for both children and adults with sickle cell anemia. However, there is substantial inter-patient variability with regard to the optimal dosing regimen, as well as differences in treatment-related toxicities and responses that may be explained by hydroxyurea pharmacokinetics and pharmacogenetics. Addressing the safety and efficacy of hydroxyurea treatment requires quantitative and accurate drug analysis, and various laboratory techniques have been established. We review the historical and current analytical techniques for measuring hydroxyurea concentrations accurately, and discuss clinical settings where quantitative analysis can increase understanding and safety of this important therapeutic agent, and ultimately improve patient outcomes.

Background: Hydroxyurea is now the standard of care for children with sickle cell anemia (SCA). Results from the BABY HUG study and recommendations from the 2014 NHLBI Guidelines have led to early initiation (increasingly before 1 year of age) of hydroxyurea for many patients. Given the known variability in hydroxyurea pharmacokinetics (PK), treatment response (HbF%), and maximum tolerated dose (MTD), we hypothesized that individualized dosing would provide the optimal treatment approach. Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154) is a prospective study of a personalized, PK-guided dosing model of hydroxyurea for children with SCA. Using population PK model-based Bayesian estimation, each participant's PK data are used to generate an individualized starting hydroxyurea dose that targets an area under the curve associated with actual MTD. Clinical follow-up and subsequent dose adjustments target MTD, usually defined by ANC Results:The analysis of children starting hydroxyurea before 2 years of age included 33 participants (of 47 total TREAT enrollments), who contributed a total of 59.5 patient-years of hydroxyurea therapy. The mean age (±SD) at hydroxyurea initiation was 1.0±0.4 years of age. The average PK-guided, individualized starting dose was 27.8±5.3 mg/kg/day, higher than conventional and BABY HUG initial dosing (20 mg/kg/day). For children who have completed 12 months of therapy (n=24), effects in hematologic laboratory data are remarkable with average 35.9±8.9% HbF and hemoglobin concentration of 10.2±1.1 g/dL after 12 months of therapy (compared to 29.3±8.8% and 9.2±1.3 g/dL at baseline). The majority (70%) of these participants have HbF>30% and almost half achieved HbF>40% after 12 months of hydroxyurea. This hematological response is more robust than what was observed in BABY HUG (HbF=22.4%, Hb=9.1 g/dL after two years of therapy, Wang WC et al. Lancet 2011). In the TREAT cohort, there were no episodes of dactylitis, acute splenic sequestration, or stroke. There were 111 emergency room or sick outpatient clinic visits for this young cohort; 107 ED/clinic visits (without subsequent hospitalization) were for fever, URI symptoms, GI illness, or other non-specific complaints unrelated to SCA, while only 4 (3.6%) visits were for pain. There were 38 hospitalizations in 17 participants, mostly for routine evaluation of fever (66%), but no positive blood cultures and no admissions for febrile neutropenia. The average length of hospitalization was 2.8±2.4 days with 81% of participants discharged within 72 hours of admission. There were 3 episodes of acute chest syndrome in 2 patients, two of whom required PRBC transfusion. Including all types of visits, there were only 6 pain events, equivalent to 10.1 pain events per 100 patient-years, which is much lower than the published 94 events per 100 patient-years in the hydroxyurea treatment arm of BABY HUG (Thornburg CD et al. Blood 2012). There were 37 TCD exams performed in 16 participants, all normal except for one patient with conditional velocities that normalized with hydroxyurea. There were no significant differences from baseline to month 12 in either RBC pit counts or cystatin C values. Conclusions: Hydroxyurea initiation at an early age using PK-guided dosing provides significant clinical benefits for young children with sickle cell anemia. These TREAT study data suggest that initiating hydroxyurea around one year of life using a personalized dosing strategy can provide better clinical and laboratory benefits than starting at the conventional 20 mg/kg/day weight-based dose. Very high HbF levels are observed at modest and well-tolerated doses of hydroxyurea, perhaps because treatment was initiated before the process of HbF inactivation is complete. Continued long-term follow-up of these patients will determine whether these will be sustained and able to prevent both short- and long-term complications of SCA. Disclosures Malik: CSL Behring: Patents & Royalties. Quinn:Silver Lake Research Corporation: Research Funding; Global Blood Therapeutics: Research Funding; Amgen: Research Funding. Ware:Biomedomics: Research Funding; Nova Laboratories: Consultancy; Bristol Myers Squibb: Research Funding; Addmedica: Research Funding; Global Blood Therapeutics: Other: advisory board; Agios: Other: advisory board; Novartis: Membership on an entity's Board of Directors or advisory committees.

Hydroxyurea is a potent and safe disease-modifying therapy for both children and adults with sickle cell anemia (SCA). Several decades of research data have documented clinical efficacy characterized by fewer acute complications, improved organ function, and reduced mortality. Relatively sparse data exist, however, regarding the risks and benefits of hydroxyurea for mothers with SCA during pregnancy and lactation. Mothers with SCA who wish to breastfeed their babies are discouraged from taking hydroxyurea, due to mostly theoretical concerns about transfer of the drug into breast milk. Determination of the pharmacokinetics (PK) of hydroxyurea in breast milk, along with accurate measurement of the amounts transferred to infants during lactation, are warranted since hydroxyurea is now commonly prescribed to women of child-bearing years. We designed the prospective HELPS trial (Hydroxyurea Exposure in Lactation: a Pharmacokinetics Study, NCT02990598) with the following objectives: (1) determine the PK profile of hydroxyurea in blood and breast milk; (2) calculate the distribution of hydroxyurea between plasma and milk compartments; (3) measure the amount of hydroxyurea excreted into the milk and urine; and (4) create population PK profiles to estimate infant drug exposure. Lactating women were recruited to receive a single oral dose of hydroxyurea (two 500mg capsules). Breast milk, blood, and urine were collected before the dose and then at scheduled timepoints, including 15, 30, 60, 120, 180, 240, 360, 480, 720, and 1440 minutes. Hydroxyurea concentrations were measured using a validated HPLC assay. A total of 16 women (14 healthy volunteers and 2 with SCA; 11 white and 5 black) were recruited into HELPS; the median age was 31 years (range 26-38 years) and the median weight was 76.3 kg (range 48.8-115.9 kg). All women were currently lactating and breastfeeding their infants for a median duration of 6 months (range 2-22 months). After the 1000mg dose, peak plasma hydroxyurea concentrations ranged from 10-40 µg/mL, typically at 60-120 minutes after dosing although absorption varied among participants. Hydroxyurea was measured in breast milk at ~80% of the plasma concentration, with both compartments having a similar PK profile for each participant, from absorption through distribution and clearance. No hydroxyurea was detected in either plasma or breast milk 24 hours after the dose. With this frequent collection schedule, an average of 2.2 ± 1.0 mg of hydroxyurea transferred into breast milk over the 24-hour timeframe, with the majority transferred in the first 3-6 hours. Most of the drug was excreted into the urine, with an average of 542 ± 178 mg (range 295-876 mg) measured over the 24-hour timeframe; over 90% of urinary excretion occurred in the first 9 hours after the dose. The collection schedule was then modified to assess drug accumulation and equilibrium in more physiologically relevant 3-hour intervals, which reduced the average amount of hydroxyurea transfer to 1.1 mg; this 50% reduction in drug transfer indicated a rapid equilibrium between milk and plasma compartments. Two lactating women with SCA had similar hydroxyurea PK curves for plasma and milk to the 14 healthy volunteers. PK analysis based on the frequent milk collection schedule and a standard 150 mL/kg intake per 24 hours concluded that 0.41 ± 0.14 mg hydroxyurea will transfer per kg of infant weight per day. Using the World Health Organization definition of Relative Infant Dosage (RID), defined as the infant dose (mg/kg/day) compared to the maternal dose (mg/kg/day), this infant hydroxyurea exposure is 3.1%, where The HELPS trial provides definitive measurements and PK data regarding hydroxyurea in human breast milk, confirming that drug does transfer but has a rapid equilibrium between plasma and milk. Breastfeeding mothers will transfer only a small amount of hydroxyurea to their infants, so lactation during hydroxyurea therapy should not be contraindicated. These data are relevant for expanded access to hydroxyurea as a disease-modifying therapy to lactating women around the world. Disclosures Ware: Novartis: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Other: advisory board; Bristol Myers Squibb: Research Funding; Addmedica: Research Funding; Agios: Other: advisory board; Nova Laboratories: Consultancy; Biomedomics: Research Funding.

Mitochondria, also known as "Power House of cell," are crucial organelles, regulating energy metabolism. Recently, an involvement of mitochondria in cancer occurrence and metastasis has been proposed. The roles of mitochondria in cancer progression/metastasis include alteration of glycolysis, regulation of ROS and suppression of intrinsic apoptosis. This mini-review explains the specific mitochondrial characteristics during cancer metastasis with past and recent findings. It may contribute to understanding mitochondria-related mechanisms of cancer metastasis.

Hyperlipidemia is a major contributor for atherosclerosis and hypolipidemic drugs such as statin are highly prescribed to treat elevated lipid level in plasma. Rubus coreanus, which is widely cultivated in south eastern Asia, have been reported to show significant cholesterol lowering action in hyperlipidemic subjects. Our objective was to determine the cellular effect of Rubus coreanus extract (RCE) on cholesterol biosynthesis in human hepatic cells (HepG2) and to elucidate the molecular mechanism by which it causes change in cholesterol metabolism. RCE treatment lowered cholesterol biosynthesis as well as secretion from HepG2 cells. This effect was associated with lowering the release of apolipoproteins from hepatic cells. RCE treatment also showed an increase in phosphorylation of foxhead box protein 01 (FoXo-1) and 5-adenosine monophosphate-activated protein kinase (AMPK), thus lowering expression of phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase, which might be a major pathway for cholesterol biosynthesis inhibition. Apart from this; RCE also lowered sterol regulatory element-binding protein-1 (SREBP-1) expression in HepG2 cells, showing a long term regulation of cholesterol biosynthesis activity. These results indicate that one of the anti-hyperlipidemic actions of RCE is due to inhibition of cholesterol biosynthesis in hepatic cells and provides first documentation of a hypolipidemic bio-molecular action of Rubus coreanus.

Endoplasmic reticulum (ER) stress and associated protein aggregation are closely associated with human diseases, including alterations in hepatic lipid metabolism. Inhibition of ER stress can have a significant effect on the prevention of hepatic dyslipidemia. Here, we studied the role of 4-phenylbutyric acid (4-PBA), a chemical chaperone, on ER stress-induced hepatic lipid accumulation. We studied ER stress induction following CCl4 exposure and delineated mechanisms of the CCl4-induced ER stress response in liver tissue from mice. CCl4 affected the formation of disulfide bonds through excessive hyper-oxidation of protein disulfide isomerase (PDI). Increased complex formation between PDI and its client proteins persisted in CCl4-exposed samples. Conversely, 4-PBA inhibited ER stress via secretion of apolipoprotein B and prevention of hepatic lipid accumulation. We also studied the mechanism-based pharmacokinetic and pharmacodynamic profiles and identified the ER stress-related proteins GRP78 and CHOP, along with plasma apolipoprotein B and triglyceride levels, as novel biomarkers of ER stress-induced hepatic lipid accumulation. ER stress and its clinical relevance for therapeutic approaches were well correlated with the activity of the ER stress regulator 4-PBA, which may be a promising drug candidate for the treatment of hepatic lipid accumulation, such as hepatic steatosis.

Hyperactivation of phosphoinositol 3-kinase (PI3K) has been suggested to be a potential mechanism for endoplasmic reticulum (ER) stress-enhanced airway hyperresponsiveness, and PI3K inhibitors have been examined as asthma therapeutics. However, the regulatory mechanism linking PI3K to ER stress and related pathological signals in asthma have not been defined. To elucidate these pathogenic pathways, we investigated the influence of a selective PI3Kδ inhibitor, IC87114, on airway inflammation in an ovalbumin/lipopolysaccharide (OVA/LPS)-induced asthma model. In OVA/LPS-induced asthmatic mice, the activity of PI3K, downstream phosphorylation of AKT and activation of nuclear factor-κB (NF-κB) were all significantly elevated; these effects were reversed by IC87114. IC87114 treatment also reduced the OVA/LPS-induced ER stress response by enhancing the intra-ER oxidative folding status through suppression of protein disulfide isomerase activity, ER-associated reactive oxygen species (ROS) accumulation and NOX4 activity. Furthermore, inositol-requiring enzyme-1α (IRE1α)-dependent degradation (RIDD) of IRE1α was reduced by IC87114, resulting in a decreased release of proinflammatory cytokines from bronchial epithelial cells. These results suggest that PI3Kδ may induce severe airway inflammation and hyperresponsiveness by activating NF-κB signaling through ER-associated ROS and RIDD-RIG-I activation. The PI3Kδ inhibitor IC87114 is a potential therapeutic agent against neutrophil-dominant asthma.

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

Background: Hydroxyurea is the primary disease modifying therapy for patients with sickle cell anemia (SCA). The clinical and laboratory benefits of hydroxyurea are the greatest when escalated to the maximum tolerated dose (MTD). The process of dose escalation to MTD requires expertise and can be tedious, often taking 6-12 months to titrate to the optimal dose. In addition, due in part to inter-patient variability in hydroxyurea pharmacokinetics (PK), the MTD varies among patients with a range of 15-35 mg/kg/day. We utilized a population PK model in combination with Bayesian estimation and a sparse sampling strategy, to individualize dosing of children starting hydroxyurea treatment. Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154) is a prospective study of hydroxyurea for children with SCA. The primary objective is to develop and evaluate a population PK-based model to predict hydroxyurea MTD through an individualized dosing strategy. A sparse sampling approach was developed to allow practical sampling from young children with SCA. The sampling strategy includes administering a single oral 20 mg/kg dose followed by collection of small quantities of blood (~100uL) at three post-dosing time points (15-20 minutes, 50-60 minutes, and ~3 hours). Baseline labs (including liver and renal function) are typically collected by venipuncture, while the other two samples are drawn by fingerstick or heelstick. Plasma hydroxyurea concentrations are measured using HPLC using an internal standard of methylurea. Using the population PK model with Bayesian estimation and hydroxyurea concentrations measured at the three specified time points, hydroxyurea exposure is estimated using specialized therapeutic drug monitoring software (MWPharm, Mediware, Prague, Czech Republic). Using the area under the curve (AUC0-inf) estimated by the model, we calculate a starting dose that is predicted to achieve an AUC of 115 ug*h/mL, which was the mean AUC value at MTD for a large cohort of children from a previous study (Dong M et al. Br J Clin Pharmacol 2016). The primary objective is to select a starting dose that is close to actual MTD, to reduce the time to maximum therapeutic effect and need for dose modifications before achieving MTD. Results: From December 2014 through June 2016, 20 children taking taking hydroxyurea for the first time were enrolled in TREAT. Seventeen of the 20 participants had all 3 post-treatment PK samples collected and processed to allow calculation of an individualized PK-based dose, while 3 had difficulties in sampling or processing that prevented a safe PK-guided dose recommendation. These 3 participants were started at the standard hydroxyurea dose of 20 mg/kg/day. The Table summarizes baseline characteristics for the initial study population, notable for a very young starting age with 13/20 (65%) less than two years of age. Twelve children with PK-based initial dosing have been treated with hydroxyurea for at least six months. Despite the young starting age, after six months of hydroxyurea, children have documented increases in total Hb (1.4+/-1.9 g/dL), HbF (11.3+/-6.4%), and MCV (15+/-8 fL) and decreases in absolute reticulocyte count (-217+/-128 x 109/L) and absolute neutrophil count (-1.0+/-1.9 x 109/L). In 9 of 12 participants, the PK-guided initial dose remained the best clinical dose at six months without significant dose changes except for minor adjustments for weight. Two patients required a single dose escalation due to inadequate marrow suppression, while one required a dose hold and decrease due to neutropenia during and following a viral infection. Conclusions: These data demonstrate that a sparse sampling approach, requiring only 3 blood samples over 3 hours, is able to accurately estimate hydroxyurea exposure in children with SCA. Hydroxyurea exposure, as defined by AUC, was similar with this sparse sampling approach as previous studies that relied upon a more standard and prolonged PK sampling approach. This population PK model is then able to predict a safe starting dose of hydroxyurea that approximates the actual MTD, with clinically significant improvements in laboratory parameters following six months of therapy. This individualized PK-guided dosing regimen should simplify hydroxyurea dosing and reduce the time interval to reach MTD and maximal clinical benefits. Table Table. Disclosures Kalfa: Baxter/Baxalta/Shire: Research Funding. Quinn:Silver Lake Research Corporation: Consultancy; Amgen: Research Funding; Eli Lilly: Research Funding. Ware:Nova Laboratories: Consultancy; Addmedica: Research Funding; Global Blood Therapeutics: Consultancy; Bayer Pharmaceuticals: Consultancy; Biomedomics: Research Funding; Bristol Myers Squibb: Research Funding.

IC87114 is a selective PI3Kδ inhibitor. A simple, sensitive and reliable LC–MS/MS method with rapid sample preparation was developed and validated for the determination of IC87114 in mouse plasma, bronchoalveolar lavage, and lung. Chromatographic separation was achieved using an Agilent Zorbax Eclipse XDB-C 18 column (150 mm × 2.1 mm internal diameter, 3.5 μm particle size). Mass spectrometric detection was conducted by electrospray ionization in positive ion multiple reaction monitoring modes. The calibration curve was linear over a concentration range of 0.01–1000 ng/mL for plasma/BAL and 0.1–250 ng/mL for lung tissue. Recoveries were as high as 97.29%, 102.81% and 89.70% for plasma, BAL fluid and lung sample, respectively. The lower limit of quantification was 0.01 ng/mL. Intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. Finally, the method was successfully used in a pharmacokinetic study that measured IC87114 in mouse plasma, BAL fluid and lung tissue after administration of a single 1 mg/kg intratracheal dose of IC87114. The percentage change for incurred sample reanalysis (ISR) was within ±15.0% and met the acceptance criteria for ISR.

Hyperlipidemia is a major contributor for atherosclerosis and hypolipidemic drugs such as statin are highly prescribed to treat elevated lipid level in plasma. Rubus coreanus, which is widely cultivated in south eastern Asia, have been reported to show significant cholesterol lowering action in hyperlipidemic subjects. Our objective was to determine the cellular effect of Rubus coreanus extract (RCE) on cholesterol biosynthesis in human hepatic cells (HepG2) and to elucidate the molecular mechanism by which it causes change in cholesterol metabolism. RCE treatment lowered cholesterol biosynthesis as well as secretion from HepG2 cells. This effect was associated with lowering the release of apolipoproteins from hepatic cells. RCE treatment also showed an increase in phosphorylation of foxhead box protein 01 (FoXo-1) and 5-adenosine monophosphate-activated protein kinase (AMPK), thus lowering expression of phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase, which might be a major pathway for cholesterol biosynthesis inhibition. Apart from this; RCE also lowered sterol regulatory element-binding protein-1 (SREBP-1) expression in HepG2 cells, showing a long term regulation of cholesterol biosynthesis activity. These results indicate that one of the anti-hyperlipidemic actions of RCE is due to inhibition of cholesterol biosynthesis in hepatic cells and provides first documentation of a hypolipidemic bio-molecular action of Rubus coreanus.

Background: Hydroxyurea is currently the only US FDA-approved treatment for patients with sickle cell anemia (SCA), and 30 years of research have documented its safety and efficacy for this life-threatening disorder. However, there is surprisingly little known about the tissue distribution and drug clearance of hydroxyurea in humans, and the influence of drug pharmacokinetics (PK) on the hematological responses and toxicities of hydroxyurea treatment. In 2014, the National Heart Lung and Blood Institute published evidence-based guidelines that recommend wider use of hydroxyurea for adults and children with SCA, even offering therapy to infants. Accordingly, it is critically important to understand the PK parameters of hydroxyurea and to elucidate its biological tissue distribution, to better understand its benefits and risks for patients with SCA. Methods: C57BL/6 mice, age 8 to 10 weeks, received a single 100 mg/kg dose of hydroxyurea by either intraperitoneal (IP) or oral gavage (OG) route, since this dosage causes myelosuppression and hematological toxicity when administered on a daily basis. After drug administration, plasma and tissue samples were collected serially at multiple time points, with samples collected and analyzed from ≥5 different mice at each time point. Hydroxyurea concentrations in plasma and tissues were analyzed by high performance liquid chromatography (HPLC); an internal standard (methylurea) and standard curve using the appropriate tissue homogenate were used to quantify hydroxyurea accurately to a threshold sensitivity of 5-10mM. Pharmacokinetic parameters were determined via non-compartmental analysis using Phoenix WinNonlin® (version 6.2.1). Results: Both routes of hydroxyurea administration featured rapid drug absorption, distribution, and clearance (Table). The maximum plasma concentration of hydroxyurea was approximately 4-fold higher following IP dosing, but the apparent elimination half-life (t1/2) was similar for both dosing routes at 15 minutes for IP and 12 minutes for oral. Following IP administration, the highest tissue concentrations of hydroxyurea were observed after ~10 minutes, which was same as plasma, indicating very rapid tissue distribution and clearance. Hydroxyurea was most highly concentrated in the kidney and spleen, along with the lung and heart, possibly reflecting overall blood flow. Testes and brain had small amounts of hydroxyurea detected by either route, but the liver had almost no hydroxyurea detected. Hydroxyurea was completely eliminated from all organs within 2 hours. Conclusion: Using a novel, accurate, and sensitive HPLC method to quantify hydroxyurea, these data provide important information on the PK parameters and tissue distribution profile of hydroxyurea when administered by either IP or OG route in a mouse model, and likely mimic the exposure of patients with SCA to this drug. The peak concentrations of hydroxyurea were highest in the kidney, possibly reflecting predominantly renal uptake and clearance. The small amounts of hydroxyurea detectable in brain and testes are encouraging regarding potential organ toxicities. Future experiments using repeated dosing and treatment of sickle mice should provide additional insights regarding the potential toxicities of hydroxyurea exposure. Table 1. PK parameters following a single dose of hydroxyurea (100 mg/kg) by either IP or OG route. PK parameters IP route OG route Plasma PK parameters Cmax (ug/mL) 111.9 ± 10.6 28.2 ± 5.6 tmax (minutes) 9.0 ± 2.2 22.5 ± 8.6 t½ (minutes) 15.1 ± 3.7 12.1 ± 1.3 V (L/kg) 1.1 ± 0.3 2.2 ± 1.0 CL/F, mL/kg/min 57.9 ± 25.2 147.3 ± 8.1 MRT (minutes) 20.2 ± 8.4 27.3 ± 2.8 AUC (ug /mL*min) 1971 ± 773 767 ± 168 Maximum Tissue Concentration (ug/mL) Kidney 88.8 ± 47.8 27.4 ± 18.8 Spleen 73.2 ± 14.9 24.5 ± 10.0 Heart 35.7 ± 11.2 22.9 ± 4.9 Lung 52.0 ± 12.0 17.2 ± 12.2 Testes 19.5 ± 6.8 8.5 ± 5.4 Brain 6.7 ± 1.3 4.5 ± 2.4 Liver ND ND Cmax refers to the maximum plasma concentration, t½ refers to the apparently elimination half-life, tmax refers to peak concentration time, V is the volume of distribution, CL/F is the apparent clearance, MRT refers to mean residence time and AUC is the area-under-the-curve.ND = not detected Disclosures Off Label Use: Hydroxyurea use for children with sickle cell anemia.. Ware:Eli Lilly: Other: DSMB membership; Biomedomics: Research Funding; Bristol Myers Squibb: Research Funding; Bayer Pharmaceuticals: Consultancy.

Background Salvia miltiorrhiza (SM) has long been used as a traditional oriental medicine for cardiovascular disease. Accumulating evidence also indicates that SM has anti-osteoporotic effects. This study was conducted to examine the SM-induced anti-osteoporotic effect and its possible mechanisms with various doses of SM. Methods We studied Sprague-Dawley female rats aged 12 weeks, divided into six groups: sham-operated control (SHAM), OVX rats supplemented with SM (1, 3, 10 and 30 mg/kg) orally for 8 weeks. At the end of the experiment, blood samples were collected and biochemistry analysis was performed. Specimens from both tibia and liver were processed for light microscopic examination. DEXA and μ-CT analyses of the tibia were also performed. Results SM treatment significantly ameliorated the decrease in BMD and trabecular bone mass according to DEXA and trabecular bone architecture analysis of trabecular bone structural parameters by μ-CT scanning. In serum biochemical analysis, SM decreased the released TRAP-5b, an osteoclast activation marker and oxidative stress parameters including MDA and NO induced by OVX. Conclusions The preventive effect of SM was presumably due to its anti-oxidative stress partly via modulation of osteoclast maturation and number. In current study, SM appears to be a promising osteoporosis therapeutic natural product.