Concentrations Of Hydroxyurea Research Articles

5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase\u2013mitotic cells in apical root meristems of Allium cepa

Key messageInduction of biphasic interphase–mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa.Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S–M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

S. mediterranea Ataxia telangiectasia and Rad3‐related kinase (SmedATR) May be Required to Prevent DNA Replication Stress

DNA replication is an important mechanism involved in cellular processes like reproduction, repair, and regeneration. S. mediterranea, a planarian with an amazing regenerative ability, goes through continuous rounds of cell division. Consequently, DNA replication stress occurs with each cell division and must be resolved. From yeast to man, The DNA replication stress response (RSR) is evolutionarily conserved and necessary for life among organisms. The RSR is a signal transduction pathway regulated by Ataxia telangiectasia and Rad3‐related kinase (ATR) and Checkpoint 1 kinase (CHK1) to respond to replication stress, including DNA damage. However, the RSR has not yet been established in the planarian and its involvement in regeneration is currently unknown. We hypothesize that ATR may be essential in replication by preventing and resolving DNA damage. Regeneration experiments were conducted and results indicated that the regenerative ability of planaria were reduced in the presence of hydroxyurea (HU), a replication stress inducing agent. Low HU concentrations (2mM) had similar phenotypic results to the control which suggests that ATR and the RSR may be present in this low dosage to overcome the replication stress. However, when treated with a combination of 2mM HU and ATR inhibitor (ATRi), planarian regeneration was severely hindered. To confirm replication stress shown phenotypically in the regeneration experiments, comet assays were performed to quantify the levels of DNA damage in each experimental group. The ATRi and 2mM HU combination group exhibited the highest average comet tail moment, a parameter used to measure the amounts of fragmented DNA within each planarian cell. The synergistic effects of ATR in combination with HU suggest that ATR is significant in the response to replication stress during regeneration. Additionally, inhibition of CHK1, another kinase in the RSR pathway exhibited similar tail moment values as ATRi. Future experiments will allow us to support the presence of CHK1 and its important role in resolving replication stress. Determining the importance of RSR genes in planarian will elucidate the conserved components and allow us to understand how ATR and the RSR contributes to the incredible regenerative abilities in planarian.Support or Funding InformationUST Committee for Student Research Alvarado Sanchez Lab, Stowers Institute for Medical Research Jim Jenkin for S. mediterranea Department of Education HSI‐STEM grant UST Biology Smith Chair UST Biology Department

A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp.

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland’s Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

Therapeutic Potential of Nanoparticle-loaded Hydroxyurea on Proliferation of Human Breast Adenocarcinoma Cell Line.

Although Hydroxyurea is one of the most widely used drugs in treating breast cancer, the use of it leads to some side effects. Hence, in order to reduce complications of treatment and increase its efficiency, drug delivery has been attracted more attention. Present study included three stages. The first stage was involved in the synthesis of nanoparticles-loaded Hydroxyurea that its characteristics were evaluated by using scanning electron microscopy and Zetasizer system. In the second stage, cultured MCF-7 cells were undergone treatments by Hydroxyurea and Nanoparticles-loaded Hydroxyurea in various concentrations. In the third stage, the MCF-7 was treated by IC50 of Hydroxyurea and nanoparticles-loaded Hydroxyurea which are in combination with radiation and hyperthermia. Afterward, the viable of cell, apoptosis, and levels of caspase-8 and-9 proteins were assessed. The average size and the potential surface of nanoparticles and nanoparticles-loaded Hydroxyurea were 26 nm, 48 nm, 3.86 mV, and -29.3 mV, respectively. Results of MTT assay and apoptosis represented that the percentage of cytotoxicity in the treated groups by in combination group and nanoparticles-loaded Hydroxyurea was significantly increased in comparison with Hydroxyurea. This increase was dependent on the concentration of nanoparticles-loaded Hydroxyurea. Nevertheless, the activity of caspase-8 shows any significant changes, the activity of caspase-9 was significantly increased in the control and treatment groups. We concluded that nanoparticles-loaded Hydroxyurea and it in combination with radiation and hyperthermia induces mitochondrial-dependent apoptosis by down-regulation of caspase-8 and up-regulation of caspase-9 expressions and have higher toxicity effect on MCF-7 cells in comparison with pure Hydroxyurea.

Hydroxyurea Pharmacokinetic Profiles in Children Treated for Extreme Thrombocytosis after Total Pancreatectomy with Islet Cell Autotransplant Demonstrate Poor Absorption

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

The Drosophila melanogaster PIF1 Helicase Promotes Survival During Replication Stress and Processive DNA Synthesis During Double-Strand Gap Repair.

PIF1 is a 5' to 3' DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.

Development of biodegradable starch nanocrystals/gum Arabic hydrogels for controlled drug delivery and cancer therapy

The study involves preparing starch nanocrystals by acid hydrolysis of starch and to produce a new chemical crosslinking starch nanocrystals/gum Arabic blend in the presence of potassium persulfate (K2S2O8) as an initiator. The new materials were characterized and its potential utilization in controlled drug delivery and cancer therapy were investigated. Crosslinked starch nanocrystals/gum Arabic blends were loaded with hydroxyurea drug. The properties of starch nanocrystal was characterized by x-ray diffraction (XRD), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The starch nanocrystal /gum Arabic blends were analyzed in vitro drug release studies by using UV–vis spectroscopy. The swelling test and MTT assay were also investigated. The drug release and the swelling test were monitored at pH7.4 and pH4. XRD result showed that starch nanocrystal was the crystalline A-type. Starch nanocrystal showed a high degree of crystallinity compared with the native starch. The experimental results proved that starch nanocrystal/gum Arabic/hydroxyurea blends reduced the swell ability of blends which lead to slow release of hydroxyurea, and the rate of hydroxyurea release was significantly higher at pH7.4 than pH4, at constant incubation time. The swelling ratio and drug release were decreased by using starch nanocrystal/hydroxyurea while the swelling ratio and drug release using starch nanocrystal/gum Arabic/hydroxyurea blend were lowest. The results of vitro cytotoxicity study showed that the 100 μg ml−1 hydroxyurea concentration killed ∼39% colorectal cancer cells while ∼27% of cells were killed by using starch nanocrystal/gum Arabic/hydroxyurea blends at the same concentration for 24 h of incubation. It was concluded that the hydroxyurea loaded starch nanocrystal/gum Arabic blend enhanced the controlled drug release system thereby, revealing a novelsvehicle for cancer therapy.

Hydroxyurea Exposure in Lactation—a Pharmacokinetics Study (HELPS)

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 <5% is generally considered safe and acceptable. However, since 48% of the drug transfers in the first 3 hours, the RID would be 1.6% if the mother fed her baby before the dose and waited 3 hours before the next feeding, and <1% when using a single “pump and dump” process.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. DisclosuresWare: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.

Hydroxyurea (hydroxycarbamide) genotoxicity in pediatric patients with sickle cell disease.

Hydroxyurea (HU) reduces the severity of sickle cell disease (SCD) in children; nevertheless, its long-term safety is an important concern. This paper evaluates HU genotoxicity at dose≤30mg/kg/day after over 2 years of treatment. The study included 76 children: 32 SCD patients treated with HU, 27 SCD patients not treated with HU, and 17 unaffected children. HU patients were classified as good or poor responders according to their clinical response. Comet assay allows the comparison of DNA damage between both groups of patients and unaffected children. Maximal concentration (Cmax ) of HU in plasma was determined after drug administration. Mean values of DNA in the comet tail were 5.13±6.84 for unaffected children, 5.80±7.78 for patients with SCD treated with HU, and 5.61±6.91 for patients with SCD not treated with HU. Significant differences were observed between unaffected children and children with SCD. No difference was evident between comets from SCD patients treated and not treated with HU. In the case of HU, mean DNA in the comet tail was significantly lower in good responders than in poor responders: 5.54±7.77 and 6.69±8.43, respectively. Mean Cmax value on plasma was 39.08±15.65mg/l; N=31. SCD increases, slightly but significantly, DNA damage in lymphocytes from patients with SCD. Patients with SCD treated with HU do not present more nucleoid damage than patients with SCD not treated with HU. Good responders to the HU treatment have significantly less nucleoid damage than poor responders. HU treatment at ≤30mg/kg/day does not expose patients to a genotoxic plasma concentration.

Determination of hydroxyurea in human plasma by HPLC-UV using derivatization with xanthydrol.

A simple and rapid high performance liquid chromatography (HPLC) method using ultraviolet (UV) detection was developed to determine hydroxyurea (HU) concentration in plasma sample after derivatization with xanthydrol. Two hundred microliters samples were spiked with methylurea (MeU) as internal standard and proteins were precipitated by adding methanol. Derivatization of HU and MeU was immediately performed by adding 0.02M xanthydrol and 1.5M HCl in order to obtain xanthyl-derivatives of HU and MeU that can be further separated using HPLC and quantified using UV detection at 240nm. Separation was achieved using a C18 column with a mobile phase composed of 20mM ammonium acetate and acetonitrile in gradient elution mode at a flow rate of 1mL/min. The total analysis time did not exceed 18min. The method was found linear from 5 to 400μM and all validation parameters fulfilled the international requirements. Between- and within-run accuracy error ranged from -4.7% to 3.2% and precision was lower than 12.8%. This simple method requires small volume samples and can be easily implemented in most clinical laboratories to develop pharmacokinetics studies of HU and to promote its therapeutic monitoring.

Hydroxyurea: Analytical techniques and quantitative analysis

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.

ERK1/2 Activation Following Renal Injury Is Required For Kidney Injury Molecule‐1 Production

BackgroundBecause substantial damage can occur before detection of acute kidney injury (AKI) new early predictor biomarkers are needed, such as kidney injury molecule‐1 (KIM‐1). KIM‐1 is a transmembrane glycoprotein that is expressed at very low levels in healthy kidney and is highly upregulated during injury. KIM‐1 is shed during injury and can be detected in blood and urine. While KIM‐1 has been studied in various models of AKI, the regulatory mechanisms leading to KIM‐1 upregulation remains limited. Here, we explored the role of ERK1/2 activation on KIM‐1 expression following toxicant exposure to proximal tubule cells and renal ischemia reperfusion injury (IRI) and LPS‐induced sepsis in mice.MethodsMouse kidney proximal tubule cells (TK) were treated with varying concentrations of hydroxyurea (HU), hydrogen peroxide (H2O2), and tert‐butyl‐hydroperoxide (TBHP) with and without pretreatment with the MEK1/2 inhibitor trametinib (10 nM). Trametinib (1mg/kg) was administered 1 hr before renal IRI to C57Bl/6 mice and were euthanized at 3 hr and 24 hr. TLR4 KO and WT mice underwent 3 hr renal IRI or septic AKI for 18 hr using LPS (10 mg/kg) injection. Signaling pathways were explored using RT‐qPCR and immunoblot analysis and kidney function was measured using BUN and serum creatinine.ResultsHU, H2O2, and TBHP exposure to TK cells for 24 hr caused ERK1/2 phosphorylation and upregulated KIM‐1 mRNA. Pretreatment with trametinib prevented KIM‐1 up‐regulation and ERK1/2 phosphorylation at 24 hr. At 3 hr post renal IRI KIM‐1 mRNA increased 4‐fold while pretreatment with trametinib resulted in a 2‐fold increase in KIM‐1 mRNA. Serum creatinine increased 4‐fold 3 hr after IRI and was attenuated by trametinib pretreatment. Cortical KIM‐1 mRNA increased 600‐fold 24 hr after IRI and was reduced 66% using trametinib in IRI. KIM‐1 protein was observed at 24 hr but not at 3 hr post IRI. Trametinib decreased KIM‐1 protein in the kidney 24 hr post IRI. TLR4 KO mice subjected to IRI exhibited increased KIM‐1 mRNA and ERK1/2 phosphorylation after 3 hr. In contrast, TLR4 KO mice exposed to LPS did not exhibit increased KIM‐1 mRNA or protein, nor activated ERK1/2 after 18 hr. WT mice treated with LPS had increased KIM‐1 mRNA and protein and ERK1/2 phosphorylation.ConclusionWe have linked pathophysiological ERK1/2 phosphorylation to the regulation of KIM‐1 within the kidney cortex. ERK1/2 phosphorylation and KIM‐1 mRNA increased following toxicant exposure in TK cells, IRI in mice, and LPS in mice, and trametinib attenuated these increases. LPS‐induced AKI required TLR4 for ERK1/2 activation and subsequent upregulation of KIM‐1. In contrast, IRI mice did not require TLR4 for ERK1/2 activation. These results demonstrate that ERK1/2 is a key initiator of renal injury.Support or Funding InformationGM084147 ‐ RGSF31DK105782 ‐ JBC

Individualized Dosing of Hydroxyurea for Children with Sickle Cell Anemia Using a Population Pharmacokinetic-Based Model: The TREAT Study

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. [Display omitted] DisclosuresKalfa: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.

Stable-Isotope Dilution HPLC–Electrospray Ionization Tandem Mass Spectrometry Method for Quantifying Hydroxyurea in Dried Blood Samples

Sickle cell anemia (SCA) is a life-threatening blood disorder characterized by the presence of sickle-shaped erythrocytes. Hydroxyurea is currently the only US Food and Drug Administration-approved treatment and there is a need for a convenient method to monitor compliance and hydroxyurea concentrations, especially in pediatric SCA patients. We describe a novel approach to the determination of hydroxyurea concentrations in dried whole blood collected on DMPK-C cards or volumetric absorptive microsampling (VAMS) devices. Hydroxyurea was quantified by electrospray ionization LC-MS/MS using [13C15N2]hydroxyurea as the internal standard. Calibrators were prepared in whole blood applied to DMPK-C cards or VAMS devices. Calibration curves for blood hydroxyurea measured from DMPK-C cards and VAMS devices were linear over the range 0.5-60 μg/mL. Interassay and intraassay CVs were <15% for blood collected by both methods, and the limit of detection was 5 ng/mL. Whole blood hydroxyurea was stable for up to 60 days on DMPK-C cards and VAMS devices when frozen at -20 °C or -80 °C. Whole blood hydroxyurea concentrations in samples collected on DMPK-C cards or VAMS devices from SCA patients were in close agreement. This tandem mass spectrometry method permits measurement of hydroxyurea concentrations in small volumes of dried blood applied to either DMPK-C cards or VAMS devices with comparable performance. This method for measuring hydroxyurea from dried blood permits the evaluation of therapeutic drug monitoring, individual pharmacokinetics, and medication adherence using heel/finger-prick samples from pediatric patients with SCA treated with hydroxyurea.

Pharmacokinetics and Tissue Distribution of Hydroxyurea in a Mouse Model

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 1PK parameters following a single dose of hydroxyurea (100 mg/kg) by either IP or OG route.PK parametersIP routeOG routePlasma PK parametersCmax (ug/mL)111.9 ± 10.628.2 ± 5.6tmax (minutes)9.0 ± 2.222.5 ± 8.6t½ (minutes)15.1 ± 3.712.1 ± 1.3V (L/kg)1.1 ± 0.32.2 ± 1.0CL/F, mL/kg/min57.9 ± 25.2147.3 ± 8.1MRT (minutes)20.2 ± 8.427.3 ± 2.8AUC (ug /mL*min)1971 ± 773767 ± 168Maximum Tissue Concentration (ug/mL)Kidney88.8 ± 47.827.4 ± 18.8Spleen73.2 ± 14.924.5 ± 10.0Heart35.7 ± 11.222.9 ± 4.9Lung52.0 ± 12.017.2 ± 12.2Testes19.5 ± 6.88.5 ± 5.4Brain6.7 ± 1.34.5 ± 2.4LiverNDNDCmax 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 DisclosuresOff 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.

Nano-Formulated Hydroxyurea Enhances Drug Delivery in K562 Cells

The vasculopathy of sickle cell disease (SCD), characterized by a chronic hemolytic anemia, endothelial dysfunction and recurrent vaso-occlusive events, underlies most of the clinical complications. Hydroxyurea (HU), the only FDA approved medication to treat patients with SCD, acts via the induction of fetal hemoglobin (HbF) production. Recent studies suggest that HU increases endothelial nitric oxide (NO) production and reduces adhesion molecule expression as another potential mechanism for the observed reduction in vaso-occlusive events and improved survival in SCD. Despite its benefits, HU is currently under-utilized and we hypothesize that improved targeted drug delivery may increase its usage. The use of nano-emulsions, a class of stable emulsions formed by a monolayer of phospholipids and/or biodegradable material, is a mechanism by which to enhance targeted cellular delivery of therapeutics. We hypothesized that a nano-formulation of HU would enhance drug delivery to the bone marrow and this may be a direct way of impacting HbF expression in SCD. Nano-formulated HU (NHU) was developed using a phospholipid approach and this compound was found to be biologically stable for > 6 months.Chronic myelogenous leukemia bone marrow (K562) cells were treated with a NHU or HU suspension in concentrations ranging from 25µM to 250µM over a course of 4, 5 and 6 days. Cells were harvested for RNA isolation and qPCR was performed using primers for HbF and basal cell adhesion molecule (BCAM, a cellular adhesion molecule on red and white blood cells. On day 5, when HbF expression in K562 cells peaks, there was a five-fold increase in HbF expression treated with lower concentrations of NHU compared to untreated cells and a two-fold increase in expression compared with HU treated cells. With increasing concentrations of NHU and HU, HbF expression became similar. This suggests that the use of NHU can provide increased HbF expression at a lower concentration with likely less toxicity. Treatment of K562 cells with NHU and HU resulted in decreased BCAM expression on day 6 of treatment. Treatment with NHU produced a four-fold decrease in BCAM expression compared to the same concentration of a HU suspension which supports the concept that NHU can impact gene expression at lower doses than unformulated HU. Nanoformulated HU produces a molecule which is highly stable, and able to achieve higher intracellular concentrations with increased efficacy in K562 cells. This suggests a potential novel therapeutic for patients with sickle cell disease. DisclosuresKlings:Pfizer: Consultancy; Actelion Pharmaceuticals: Research Funding.

Pharmacokinetics-Based Individualized Dosing Strategy to Predict Maximum Tolerated Dose of Hydroxyurea in Children with Sickle Cell Anemia

▪Background: Hydroxyurea is the primary disease-modifying therapy for adults and children with sickle cell anemia (SCA). Recent NHLBI guidelines include a recommendation for expanded hydroxyurea use, particularly for young children. The laboratory and clinical benefits of hydroxyurea therapy are optimized when escalated to maximum tolerated dose (MTD), but the process of dose escalation requires expertise and frequent laboratory tests. The time to reach MTD using traditional empirical escalation usually takes >6 months, which can delay the laboratory and clinical benefits. In addition, all children with good adherence at MTD will respond to hydroxyurea, but with substantial interpatient variability in both the MTD itself and the %HbF levels achieved, suggesting important individual differences in pharmacokinetics (PK) that contribute to this phenotypic variability.Objective: The primary objective of this study was to develop an individualized Bayesian adaptive dosing strategy to reduce the time required to reach hydroxyurea MTD for children with SCA. Achieving this objective required the development of a population PK model and identification of the most informative sampling times for Bayesian estimation order to reduce the number of observations required for robust estimation of hydroxyurea PK parameters for individual patients.Methods: PK data at baseline from 96 children with SCA enrolled in the prospective Hydroxyurea Study of Long-term Effects (HUSTLE, NCT00305175) were used to develop a population PK model using nonlinear mixed effects modeling (NONMEM 7.2). Patient demographics and clinical chemistry measurements were included for covariate analyses. The final model was validated by bootstrap analysis and visual predictive check. To identify the optimal sampling times and number of samples required to robustly estimate individual PK parameters and total hydroxyurea exposure (AUC), a D-optimal design analysis was performed using the final PK model with constraints of clinical feasibility.Results: Hydroxyurea PK profiles were best described by a one compartment model with Michaelis Menten elimination and a transit absorption model. Hydroxyurea serum concentrations showed substantial interpatient variability with AUC on Day 1 ranging from 40.0 to 149.2 mg*h/L. The average AUC at MTD (mean ± SD) was 115.7 ± 34.0 mg*h/L, so this value was chosen as the target hydroxyurea AUC for the final PK model-based approach. Of the tested covariates, body weight and cystatin C were identified as significant predictors of hydroxyurea clearance, but neither serum creatinine nor estimated creatinine clearance was identified as predictors of hydroxyurea clearance. D-optimal design indicated that three serum concentrations collected at 15-20 minutes, 50-60 minutes, and 3 hours after oral administration would accurately estimate systemic hydroxyurea exposure. Figure 1 demonstrates an example from a patient, demonstrating the PK profile obtained using the sparse sampling technique (Panel A) and the modeling to predict a dose that would target a AUC of 115 mg*h/L (Panel B).Conclusions: We have established a PK model-based individualized dosing strategy to predict hydroxyurea MTD in children with SCA. Our selective sampling strategy requires only three serum samples to be collected over 3 hours and is therefore more feasible and practical, particularly for very young children, than traditional hydroxyurea PK analysis that requires frequent blood collections over 6-8 hours. This novel Bayesian approach is being prospectively evaluated in the Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154). In TREAT, hydroxyurea concentrations are measured using HPLC after a single oral dose of 20 mg/kg, requiring only 3 fingerstick blood samples over 3 hours for accurate assessment of each patient's unique hydroxyurea PK profile. The predicted MTD is then calculated based on the amount of drug required to meet the target AUC. This strategy has the potential to individualize therapy and optimize the dose titration of hydroxyurea therapy for children with SCA, such that the laboratory and clinical benefits at MTD are achieved more quickly. [Display omitted] DisclosuresOff Label Use: Hydroxyurea is not FDA-approved for children with sickle cell anemia. Ware:Bristol Myers Squibb: Research Funding; Biomedomics: Research Funding; Eli Lilly: Other: DSMB membership; Bayer Pharmaceuticals: Consultancy.

Digitoxin and its synthetic analog MonoD have potent antiproliferative effects on lung cancer cells and potentiate the effects of hydroxyurea and paclitaxel.

Despite significant advances in the understanding of lung cancer biology, the prognosis of cancer patients remains poor. Part of the failure of anticancer therapy is due to intratumoral heterogeneity in these patients that limits the efficacy of single agents. Therefore, there is an urgent need for new anticancer drugs or drug combination regimens that possess increased activity against all cellular subtypes found within the tumor. In this study, we evaluated the in vitro antiproliferative activity of the cardiac glycosides (CGs) digitoxin and its synthetic analog MonoD on H460 lung cancer cells grown under different culture conditions. The CGs were tested alone in H460 cells under routine culture as well as in cells growing under short (24–72 h) and prolonged serum starvation (7 days) in order to evaluate the activity of drugs on cancer cells under varied degrees of proliferation. Our results showed that both CGs, and MonoD in particular, have potent antiproliferative activity at clinically relevant concentrations against cells in all the tested culture conditions. In contrast, paclitaxel, hydroxyurea and colchicine were only active in cells growing in routine culture conditions, and relatively inactive in serum-starved conditions. Importantly, both CGs were able to potentiate the effect of clinically relevant concentrations of hydroxyurea or paclitaxel in serum-starved conditions. When paclitaxel was used in combination with CGs, the highest antiproliferative effect was obtained when paclitaxel was administered first, followed by either digitoxin or MonoD. Our results indicate that CGs have potential clinical applications in translational oncology especially in combination with other drugs, and warrants further investigation of CGs in more advanced preclinical models of lung cancer.

Localization sites of nuclear envelope SUN2-like proteins in root meristem cells of Allium cepa under hydroxyurea-induced DNA replication stress

Regardless of the DNA replication stress induced by low concentration of hydroxyurea (HU), root apical meristem cells of Allium cepa keep growing, and some of them override the DNA damage checkpoint mechanisms initiating either premature or an abnormal mitotic chromosome condensation. Prolonged incubation of onion seedlings with HU results in an increased level of immunodetectable proteins sharing epitopes with SUN2, one of the highly conserved elements linking nuclear envelope (NE) to the cyto- and nucleoskeletal structures. In addition to NE, phragmoplast and cell plate, our observations extend an array of subcellular compartments at which SUN2-like proteins (SUN2-LPs) are localized. These include cortical preprophase band of microtubules, centromeric regions of ana- and telophase chromosomes, and nuclear bodies (SUN2-NBs) polarly localized in interphase nuclei according to Rabl’s configuration. SUN2-NBs (distinct from fibrillarin-rich Cajal bodies) colocalize with late-replicating areas of heterochromatin and are thought to represent clustered centromeres. Three-dimensional spatial analysis of SUN2-NBs suggests their connections with NE. An enhanced expression and additional localization sites of SUN2-LPs may be correlated with a considerable reprogramming of cellular functions triggered in response to prolonged HU treatment.

Anodic voltammetric behavior of hydroxyurea and its electroanalytical determination in pharmaceutical dosage form and urine

A simple and a novel electroanalysis of hydroxyurea (HU) drug at pencil graphite electrode (PGE) has been investigated by using cyclic, linear sweep and differential pulse voltammetric techniques. The oxidation of HU was irreversible and exhibits a diffusion controlled process and is of pH dependence. The oxidation mechanism was proposed. The dependence of the current on pH, the concentration, nature of buffer, and scan rate was investigated to optimize the experimental conditions for the determination of HU. It was found that the optimum buffer for the determination of HU was pH of 8.0. In the range of 0.01 to 1.0mM, the current measured by differential pulse voltammetry presents a good linear property as a function of the concentration of HU with a limit of detection of 7.89μM. The developed method was successfully applied to HU determination in pharmaceutical formulation and human urine.