Concentrations Of Hydroxyurea Research Articles

Oxidative replication stress induced by long-term exposure to hydroxyurea in root meristem cells of Vicia faba.

Low concentrations of hydroxyurea, an inhibitor of DNA replication, induced oxidative and replicative stress in root apical meristem (RAM) cells of Vicia faba. Plant cells are constantly exposed to low-level endogenous stress factors that can affect DNA replication and lead to DNA damage. Long-term treatments of Vicia faba root apical meristems (RAMs) with HU leads to the appearance of atypical cells with intranuclear asynchrony. This rare form of abnormality was manifested by a gradual condensation of chromatin, from interphase to mitosis (so-called IM cells). Moreover, HU-treated root cells revealed abnormal chromosome structure, persisting DNA replication, and elevated levels of intracellular hydrogen peroxide (H2O2) and superoxide anion (O2∙-). Immunocytochemical studies have shown an increased number of fluorescent foci of H3 histones acetylated at lysine 56 (H3K56Ac; canonically connected with the DNA replication process). We show that continuous 3-day exposure to low concentrations (0.75mM) of hydroxyurea (HU; an inhibitor of DNA replication) induces cellular response to reactive oxygen species and to DNA replication stress conditions.

A Novel, Rapid, and Accurate Quantitative Hydroxyurea Assay

Introduction: Hydroxyurea is a highly effective treatment for children and adults with sickle cell anemia (SCA) and is now considered standard-of-care therapy in high-resource countries. There is wide interpatient variability in the optimal dose, which is defined as the daily dose that provides maximal induction of protective fetal hemoglobin to decrease erythrocyte sickling while causing only mild marrow suppression. The time and cost of hydroxyurea dose titration are barriers to its widespread use in low-resource settings. The titration process required to identify an optimal dose can be abbreviated by measuring individualized hydroxyurea pharmacokinetics (PK) to guide personalized precision dosing, which could be especially useful in low-resources settings with limited laboratory monitoring. However, measurement of hydroxyurea in serum and other physiological fluids is not commonly performed during routine clinical treatment. Current methodology to quantify hydroxyurea concentrations requires complex and time-consuming chemical derivatization followed by detection by colorimetric spectrophotometry, high performance liquid chromatography (HPLC), or liquid chromatography mass spectrometry (LC-MS). To consider performing PK-guided dosing in low-resource settings, a simple, accurate, and efficient hydroxyurea assay is needed. Methods: A commercially available open-system HPLC unit for hemoglobin separation (SmartLifeLC, PolyLC Inc, Columbia MD) was equipped with a 3.5-micron, 4.6 mm × 50 mm Zorbax Eclipse XDB-C18 column with matching guard column (Agilent Technologies, Santa Clara, CA); mobile phases of 46% methanol and 100% acetonitrile; and a 200-600nm multi-wavelength detector. The chemical derivatization process was modified from previously published techniques that use xanthydrol (Sigma-Aldrich, St. Louis MO) for detection of primary amides. N-methylurea (Sigma-Aldrich) was included as an internal standard. Hydroxyurea (Sigma-Aldrich) was diluted in whole blood collected from healthy controls to create a calibration curve encompassing the typical pharmacological range of hydroxyurea at 80, 40, 20, 10, and 5 mg/mL. Quality Control (QC) samples were prepared at 50, 25, 12.5, and 6.25 mg/mL to validate the calibration curve before testing patient samples. Blood was applied to 20mL volumetric absorptive microsampling devices (Mitra, Trajan Scientific and Medical, Torrance CA) and dried for future elution and analysis. HPLC results were compared to LC-MS results to assess accuracy. Results: Blood was eluted from the Mitra microsampling device and derivatized in a xanthydrol solution before HPLC analysis. Using a 10-minute run with 210nm wavelength detection, the xanthydrol-modified hydroxyurea reproducibly yielded a linear calibration curve with r 2 >0.95 (Figure), and the actual concentrations were within 20% of the theoretical values. Repeated testing of control and patient samples revealed a typical intra-day coefficient of variation <15% and inter-day coefficient of variation <20%. The agreement between nominal values and assay results determined for a wide range of QC samples from both methods exceeded the FDA-recommended threshold of 67% for chromatographic assay validation with 86% agreement on LC-MS and 85% with the new method. The entire elution and derivatization procedure could be completed using dried Mitra blood samples in less than 60 minutes. Conclusions: Quantitative hydroxyurea measurement of blood or serum can now be accomplished using a novel, rapid, HPLC technique that features xanthydrol-based derivatization of hydroxyurea, an internal standard, and UV wavelength detection. This technique is accurate, reproducible, and relatively simple, and thus deemed suitable for hydroxyurea concentration measurements needed for PK-guided dosing calculations for children with SCA living in low-resource settings. This novel technique will be added to the multi-country Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) trial that will feature PK-guided precision dosing for young children with SCA starting hydroxyurea treatment.

Assessment of Novel Erythroid Signaling Pathways Modulating Hydroxyurea-Mediated Reduction of Vaso-Occlusive Adhesive Interactions in Early Precursors of Sickle Red Blood Cells (RBCs)

Sickle cell disease (SCD) affects ~100,000 Americans and millions worldwide, with healthcare costs in the U.S. exceeding $1 billion annually to treat frequent and unpredictable vaso-occlusive episodes (VOEs). In addition to fetal hemoglobin induction, hydroxyurea (HU) reduces the frequency of VOEs by increasing nitric oxide (NO) bioavailability and decreasing adhesion receptor expression and RBC-endothelial interactions. Very late antigen-4 (VLA-4), the best characterized adhesion receptor in SCD, is highly expressed on circulating reticulocytes (immature RBCs) and white blood cells (WBCs) during VOEs and in SCD patients with severe disease phenotypes and, decreased in response to HU therapy. Like other integrins, VLA-4 is functionally regulated by cell signaling pathways to modulate activity and binding affinity to a wide variety of ligands that are elevated in the SCD micro-environment. There is highly intriguing evidence showing HU modulates adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity however specific mechanisms remain unclear. The objective of this study was to assess rapid cell signaling pathways involved in HU-mediated reduction in VLA-4 binding to endothelial vascular cell adhesion molecule-1 (VCAM-1). Whole blood (WB) was obtained from patients with SCD at benign clinic visits according to IRB-approved protocols. WB density-gradient centrifugation was used to isolate hematopoietic stem cells (HSCs) and WBCs from the buffy coat layer and, a CD71 monoclonal antibody (Ab) coupled with magnetic beads to isolate reticulocytes from the packed RBC layer. HSCs were matured to erythroblasts (EBs) in appropriate cell culture media and flow cytometric analyses assessed purity of blood samples using CD235a (exclusive WBC marker) and CD45 (exclusive RBC marker) Abs . Blood samples were pretreated with and without a therapeutically relevant hydroxyurea concentration (50mcM) followed by perfusion through micro-fluidic channels coated with VCAM-1 during pulsatile, shear flow (1.67Hz, 1.0 dyne/cm 2). Additionally, EBs were treated with HU combined and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one (ODQ; NO-dependent guanylate cyclase inhibitor). Adhered cells were quantified to establish an adhesion index. The non-parametric Wilcoxon matched pairs signed rank test was used to test the statistical differences between groups. Data are presented as mean ± standard error of mean. A p-value < 0.05 was considered statistically significant. There was a significant decrease in WB (p=0.01) and reticulocyte (p<0.0001) adhesion to VCAM-1 in HU-treated samples however, there was no effect on WBC (p>0.99) adhesion suggesting reticulocytes reduce the adhesive burden of blood cells in WB adhesion assays and modulation of VLA-4 binding differs in sickle RBCs when compared to WBCs. More recently, preliminary data from my lab demonstrated that EBs pretreated with HU reduces VLA-4 binding to VCAM-1 (from 126.5 ± 29.42 to 109.5 ± 29.09) by cGMP-dependent mechanisms (HU + ODQ; from 109.5 ± 29.09 to 120.2 ± 42.80). Laboratory-based static and flow adhesion assays have been used for decades to describe adhesion in SCD since earlier studies established that sickle RBCs are more adherent than non-sickle blood cells and, adhesion correlates with SCD severity. For example, the adhesion assay described above has been used to demonstrate that VLA-4 binding can stratify SCD patients based on disease severity and predict impending VOEs. Additionally, reticulocyte and HbF levels strongly correlate with VLA-4 binding to VCAM-1 and, VLA-4-mediated adhesion is increased during patient-reported VOEs and decreased in HU-treated SCD patients. This is the first SCD study demonstrating the role of HU in modulating VLA-4 binding to endothelial VCAM-1 through rapid erythroid signaling pathways. There is also compelling evidence involving downregulation of VLA-4 activity/binding affinity through NO/cGMP dependent mechanisms and enhancement of VLA-4 binding by kinase-dependent pathways. HU has also been shown to modulate adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity. Collectively, these data suggest that VLA-4 mechanisms may be targeted to therapeutically inhibit vaso-occlusive adhesive events that promote VOEs in SCD.

Pharmacokinetic-Guided Hydroxyurea to Reduce Transfusion Requirements in Ugandan Children with Sickle Cell Anemia: Baseline Data from the Alternative Dosing and Prevention of Transfusions (ADAPT) Trial

Background: Children are the primary recipients of blood transfusions in sub-Saharan Africa where the demand for safe blood far exceeds the supply. Without disease-modifying therapy, sickle cell anemia (SCA) causes excess morbidity, mortality, and healthcare resource utilization, specifically requiring a high reliance on blood transfusions. Hydroxyurea is a safe and effective medication for the treatment of SCA that decreases blood transfusion use in this population, which may offset the costs of improving hydroxyurea access. The widespread use of hydroxyurea in Africa is limited in part by challenges determining an individual's optimal dose; pharmacokinetic (PK) dosing of hydroxyurea may streamline the dosing process, maximizing the clinical benefits and minimizing toxicities. We designed the Alternative Dosing And Prevention of Transfusions (ADAPT) trial to analyze the reduction in transfusion use with hydroxyurea and as a pilot evaluation of the feasibility of PK-guided hydroxyurea dosing in Uganda. Methods: ADAPT (NCT05662098) is a prospective, single-center, open-label cohort study at Jinja Regional Referral Hospital in Uganda. Children 1-10 years old with HbSS were eligible to enroll; those already receiving hydroxyurea or scheduled blood transfusions, or with malignancy, HIV, tuberculosis, or other chronic illnesses were excluded, and those who received a transfusion within 30 days were temporarily excluded. All participants completed a 3-month screening period during which they underwent baseline evaluations and hydroxyurea PK testing. For PK testing, a 500 mg hydroxyurea test dose was administered, and serum was collected 30, 60, and 180 minutes after the dose. Hydroxyurea concentrations were determined via chemical derivatization and high-performance liquid chromatography in Uganda. Area-under-time-concentration-curves (AUC) and Bayesian estimation were then used to calculate the optimal hydroxyurea dose based on target medication exposure. Those for whom hydroxyurea PK evaluation was completed successfully with a dose recommendation between 15-35 mg/kg/day started on their PK-guided hydroxyurea dose; otherwise, participants started on the default dose of 20 mg/kg/day. All participants will be treated on hydroxyurea for a minimum of 12 months during which time both treatment groups will follow the same laboratory monitoring and dose adjustment protocol to achieve the maximum tolerated dose. The primary study endpoint is the incidence rate ratio of blood transfusion use during screening versus treatment. Secondary endpoints include characterization of blood transfusions (indications, risk factors, adverse reactions), feasibility of PK testing, and laboratory and clinical adverse events comparing children started on PK-guided versus default dosing. Results: From June 2022 to January 2023, 106 participants were enrolled, exceeding the projected enrollment pace and goal. The average age (mean ± SD) at enrollment was 4.6 ± 2.3 years, and 50% were female. Per family-reported history, most participants had previously received a blood transfusion (78%) with 15% reporting at least 6 lifetime transfusions. Five percent had a prior stroke. A majority also had a history of pain events (86%), malaria (79%), and dactylitis (74%). Only 2% had previously used hydroxyurea. At enrollment, the median weight-for-height z-score was -0.19 (interquartile range -1.03-0.32), and baseline exam demonstrated a palpable spleen in 20%. Abdominal ultrasonography was completed on all participants, and the spleen was identified in 75% of participants with a mean volume of 132 ± 99 mL. Transcranial Doppler exams were completed on 105 participants (99%) during screening with 13% conditional and 6% abnormal results. Baseline labs (mean ± SD) documented hemoglobin = 7.7 ± 1.3 g/dL and HbF = 10.3 ± 7.1 %. Of 106 children enrolled, 1 died from acute chest syndrome in screening and 2 were withdrawn after moving away from the clinic; 103 successfully initiated hydroxyurea treatment. Conclusion: ADAPT has fully completed enrollment and will prospectively evaluate the effectiveness of hydroxyurea treatment on reducing transfusion use in Ugandan children with SCA, a finding with both individual and public health implications. ADAPT will also provide pilot data regarding the feasibility of PK-guided dosing of hydroxyurea to facilitate safe, effective hydroxyurea use in Africa.

Hydroxyurea Pharmacokinetics and Fetal Hemoglobin Response in Ugandan Children with Sickle Cell Anemia

Introduction Over 300,000 infants are born with sickle cell anemia (SCA) in Africa every year, and the under-5 mortality of this population is estimated to be 50-90% without treatment. Hydroxyurea is a safe, oral medication that decreases morbidity and mortality in SCA by increasing protective fetal hemoglobin (HbF) production and reducing inflammation. Hydroxyurea dosing to maximum tolerated dose (MTD) offers the optimal treatment with greatest laboratory responses and clinical benefits. Patients have variable hydroxyurea absorption and elimination rates, so the traditional approach to determining MTD is through incremental dose escalation to achieve mild myelosuppression and reduction in absolute neutrophil count (ANC). In the United States, pharmacokinetic (PK)-guided dosing of hydroxyurea for children with SCA has been shown to decrease time to MTD and to augment treatment responses, with most patients achieving over 30% HbF. Maximizing clinical benefits and minimizing logistical burdens of dosing hydroxyurea would be advantageous for areas with high SCA prevalence, but PK parameters of African children with SCA who might have higher rates of malnutrition, infections, and organ damage that affect drug absorption, distribution, and clearance are yet to be determined. Here we describe hydroxyurea PK exposure-response in Ugandan children with SCA from the Optimizing Hydroxyurea Therapy in Children with Sickle Cell Anemia In Malaria Endemic Areas: The NOHARM MTD Study (NOHARM MTD, NCT03128515). Methods NOHARM MTD was a randomized controlled trial that compared dosing strategies between hydroxyurea at fixed, weight-based dosing (~20 mg/kg/day) and dose escalation to MTD in children with SCA living in Uganda. NOHARM MTD participants underwent PK testing after 18 months of treatment. Children were administered their prescribed hydroxyurea dose in the morning at clinic, and blood was obtained by fingerstick using Mitra® microsampler devices after 20, 60, and 180 minutes. The microsampler devices were frozen at -80C in Uganda and then transported to Cincinnati Children's Hospital for analysis of hydroxyurea concentration via liquid chromatography with tandem mass spectrometry. Using our previously published population PK model, individual hydroxyurea exposures expressed as area-under-the-curve (AUC) were generated by Bayesian estimation using the PK modeling software NONMEM (Version 7.5, ICON Development Solutions, Ellicott City, USA). A hydroxyurea exposure-response analysis was completed with HbF% as the primary pharmacodynamic outcome. Results A total of 90 NOHARM MTD participants completed PK testing; samples from 12 were excluded from full analysis due to an elevated baseline concentration suggesting the participant already received the daily hydroxyurea dose before PK testing (9), supraphysiologic concentration (1), or mislabeling (2). PK profiles were analyzed for the remaining 78 participants (mean age 6.5 years, SD 0.9); of these, 41 were on fixed dose hydroxyurea and 37 were at MTD with mean doses of 18.7 mg/kg/day (SD 1.6) and 28.3 mg/kg/day (SD 4.3), respectively (Table). Those at MTD had a higher AUC with a significantly higher %HbF (mean 19% HbF on fixed dose vs 29% at MTD, p=0.0002) and lower ANC (mean 4.7 x109/L at fixed dose vs 3.2 at MTD, p=0.0045). Eighteen participants (23%) had malnutrition with a body mass index Z-score less than -1.0, and 12 (15%) participants had renal hyperfiltration with an estimated glomerular filtration rate over 150 mL/min/1.73m2; neither condition was associated with differences in AUC. Conclusion This study demonstrates the feasibility of collecting hydroxyurea PK data with timed, sparse sampling in Uganda, and the exposure-response analysis confirms that higher hydroxyurea exposure in Ugandan children with SCA is associated with greater induction of protective HbF and reduction in ANC. Malnutrition and glomerular hyperfiltration did not affect hydroxyurea exposure in this cohort. Further studies of PK-guided dosing to optimize response and time to response in this population are needed. Future directions include building capacity for local analysis of hydroxyurea concentrations using HPLC technology, real-time dose calculation via a PK model-informed algorithm, and prospective evaluation of PK-guided dosing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Optimizing Dose and Limiting Laboratory Monitoring of Hydroxyurea in Africa: Design of the Promoting Utilization and Safety of Hydroxyurea Using Precision in Africa (PUSH-UP) Trial

Background: More than 90% of the global burden of sickle cell anemia (SCA) occurs in sub-Saharan Africa (SSA) where efforts to expand newborn screening are likely to reduce early mortality. With an increasing number of infants diagnosed and surviving, it is critical to expand access to disease modifying therapy with hydroxyurea (HU) to reduce the risk of SCA complications. Despite increasing evidence demonstrating safety and effectiveness of HU in SSA, access to and use of HU remains limited. In high-resource settings, HU monitoring is performed through routine complete blood counts (CBCs) to monitor for hematologic toxicity and guide dose escalation. Unfortunately, in most SSA settings, access to CBCs is limited due to combination of lack of laboratory equipment, insufficient supply of reagents, or excess cost to the patient or healthcare system. The lack of capacity to perform a CBC is an insurmountable barrier in many SSA settings resulting in lack of access to this highly effective and lifesaving medication. There are some who argue that lower doses (10-15 mg/kg/day) in places where laboratory monitoring may not be readily available may reduce the theoretical risk of "toxicity." While this low dose strategy is likely to have some clinical benefit compared to no treatment at all, there is now sufficient evidence to demonstrate that higher doses of HU result in more significant clinical benefits without evidence that lower dosing prevents toxicity. In the US, we have recently developed and validated an individualized, pharmacokinetics (PK)-guided dosing strategy that optimizes the starting dose immediately without the need for dose escalation. The Prioritizing Utilization and Safety of Hydroxyurea Using Precision (PUSH-UP, ClinicalTrials.gov NCT05285917) trial is an NHLBI-funded clinical trial that will evaluate the utility and effectiveness of this individualized dosing strategy in addition to a limited laboratory monitoring strategy for children with SCA in Luanda, Angola. Study Design and Methods:The PUSH-UP trial is a randomized clinical trial of HU for 450 children aged 6 months through 12 years of age with SCA that will be performed at Hospital Geral dos Cajueiros, a national health reference center in Luanda, Angola. The primary aim of the study will determine the optimal dosing strategy, including a direct comparison of using an individualized, PK-guided starting dose to the more traditional, weight-based starting dose strategies. A second but equally important aim of the study will be to evaluate the safety of HU therapy with limited laboratory monitoring. The study will evaluate the feasibility and utility of measuring HU concentrations using a battery-powered HPLC machine (SmartLife LC™) and an automated individualized dose selection tool based on PK results. An important novel and real-world aspect of the trial is the use of only 500 mg capsules, the generic form of HU that is becoming increasingly available across many African countries. The study will utilize innovative dosing strategies, including every other day or 3-4 times per week dosing, with 500 mg capsules to achieve a safe daily dose in these young children. Following informed consent, prior to HU initiation, there will be a 3-month run-in period, followed by a 24-month treatment period. After initial dose selection and study arm assignment, there will be no dose escalations, to evaluate the most likely real-world scenarios where dose escalation is not feasible. Doses will be adjusted to account for weight-gain to maintain the same mg/kg dose throughout the study. CBCs will be performed monthly for 3 months before and 3 months after HU initiation of treatment to compare the incidence of hematologic "toxicities" and to test the hypothesis that there will be no increase in toxicity with HU treatment. After this, laboratory monitoring will be sparse, relying on clinical examination and point-of-care hemoglobin only without routine CBCs. The primary study endpoint will be SCA-related adverse events and secondary endpoints will include hematologic toxicities, infections and other non-SCA-related events to assess the safety of HU therapy with limited laboratory monitoring, and quality of life measures. Conclusions: The PUSH-UP trial will address important knowledge gaps related to hydroxyurea dosing and laboratory monitoring in low-resource settings that can be used to inform clinical guidelines in sub-Saharan Africa.

Interference on the Abbott i-STAT creatinine assay caused by hydroxyurea

Abstract Serum creatinine is an important biomarker used for estimating glomerular filtration rate (eGFR). The present study was designed after a patient at our institution had a significantly elevated creatinine result taken from an i-STAT point of care test that utilizes electrochemical detection. The patient’s creatinine from the i-STAT was 3.8 mg/dL and was not consistent with their clinical history. The patient’s previous creatinine taken 2 days prior was 1.39 mg/dL and 1.27 mg/dL seven days prior. Both previous readings were measured with the Siemens Dimension Vista 1500 using a colorimetric enzymatic creatinine method. A subsequent creatinine measured on the Siemens Dimension Vista 1500 was 1.30 mg/dL. Upon chart review, the clinical team noted that the patient is taking 500 mg of hydroxyurea daily for treatment of a myeloproliferative disorder. Review of the i-STAT package insert revealed hydroxyurea as a known interfering substance and states for every 100 µmol/L hydroxyurea in the specimen, creatinine will be increased by approximately 1.85 mg/dL. Review of literature described the positive interference caused by hydroxyurea for the i-STAT creatinine and glucose assays but did not explain the mechanism of this positive interference. In order to characterize the mechanism of hydroxyurea interference, we investigated the situation further. A pool of plasma was spiked with hydroxyurea. The spiked pool was serial diluted (x2, x4, x8, x16, x32, x64, x128) to observe the creatinine results in the presence of and absence of hydroxyurea. The dilution series was analyzed using the Vista enzymatic creatinine and the Abbott i-STAT enzymatic creatinine methods. The i-STAT creatinine results show increasing creatinine concentration from interference as the concentration of hydroxyurea increased. The dilution series suggests increasing positive interference with hydroxyurea. Positive creatinine interference with hydroxyurea was not observed when analyzed on the Vista platform. Hydroxyurea is a widely used treatment for myeloproliferative disorders and also for managing sickle cell disease. Hydroxyurea is known to cause positive interference in the i-STAT enzymatic creatine method that uses electrochemical detection, with the degree of interference correlating with the concentration of hydroxyurea. The Vista colorimetric enzymatic creatinine method was not observed to have hydroxyurea interference. While both methods employ enzymatic reagent systems the final detection approach is important. Patients undergoing hydroxyurea treatment should avoid i-STAT measurement systems for creatinine.

O-12: BUILDING LOCAL CAPACITY FOR HYDROXYUREA PHARMACOKINETICS AND PRECISION DOSING IN LOW-RESOURCE SETTINGS

Purpose: Hydroxyurea is a highly effective disease-modifying treatment for sickle cell anemia (SCA) and designated an Essential Medicine by the WHO. Increasing access to hydroxyurea for SCA in low-resource settings is advocated, but the ideal approach to dosing and monitoring is debated. Escalation to maximum tolerated dose (MTD) achieves superior benefits without additional toxicities but requires incremental dose adjustments with serial monitoring. Pharmacokinetic (PK)-guided dosing uses a test dose and sparse blood sampling to model hydroxyurea metabolism and predict a personalized optimal dose, which should require fewer clinical visits, lab assessments, and dose adjustments. Currently PK-guided dosing requires expensive, complex analyses that are unavailable in low-resource settings. A simplified method for hydroxyurea PK analysis, coupled with a user-friendly dosing algorithm, could simplify dosing and monitoring. Materials and methods: Concentrated stock solutions of reagents for chemical detection of serum hydroxyurea using high performance liquid chromatography (HPLC) were stored at -80C. On the day of analysis, hydroxyurea was serially diluted in human serum, then spiked with N-methylurea as an internal standard. After deproteination with trichloroacetic acid, a Fearon reaction to detect the urea moiety was performed with diacetylmonoxime, ferric chloride, sulfuric acid, and phosphoric acid. Thiosemicarbazide was added to minimize photolability of the colored product, which was analyzed using two commercial HPLC machines: (1) a standard benchtop Agilent HPLC system with a 449nm detector and 4.6mm x 250mm, 5 micron Zorbax Eclipse XDB-C18 column; and (2) a portable PolyLC SmartLife HPLC system with a 415nm detector and 4.6mm x 50mm, 3.5 micron Zorbax column XDB-C18 column (Figure 1). The mobile phases were acetonitrile and water. After validation in the US, the portable HPLC and chemicals were transported to Tanzania for repeat analyses. Results: A calibration curve was generated using the concentration of hydroxyurea dilutions ranging from 0mM to 1000mM, plotted against the hydroxyurea:N-methylurea ratio, and both HPLC systems yielded calibration curves with R2>0.99 (Figure 1). A separate stock of hydroxyurea was diluted to create known concentrations for analysis using the calibration curve, to confirm accuracy and precision within 10-20% of the known value. Both HPLC systems measured hydroxyurea concentrations with <10% variance from the prepared concentration. Paired analysis of samples on both machines yielded results with <15% variance. Serial measurements of a standard 100 mM concentration using the PolyLC system were precise with a 2.5% coefficient of variance. After transport to Tanzania, setup, and training, the SmartLife HPLC system was able to produce similar excellent hydroxyurea concentration calibration curves with R2>0.99. Conclusion: Improving access to hydroxyurea as an essential treatment for all children living with SCA requires an approach that eases financial and logistical barriers while optimizing safety and benefit, especially in low-resource settings. We modified a portable HPLC instrument to detect hydroxyurea, validated its precision and accuracy, and confirmed capacity building and knowledge transfer to a low-resource setting. Implementation of HPLC for measurement of serum hydroxyurea concentrations is now feasible in low resource settings using available laboratory infrastructure. PK-dosing of hydroxyurea may soon be possible, with initial treatment using a personalized optimal dose.Agilent (unmodified) and SmartLife (modified) HPLC instruments for measuring serum hydroxyurea concentrationsCalibration curve of 0-1000 µM hydroxyurea measured with SmartLife PolyLC high performance liquid chromatography system demonstrating excellent R2 value in both the US (A) and Tanzania (B). The authors do not declare any conflict of interest

Stability evaluation of compounded hydroxyurea 100 mg/mL oral liquids using a novel analytical method involving chemical derivatization.

This study assessed the stability of six extemporaneously compounded hydroxyurea oral liquids stored at room temperature. Hydroxyurea oral liquids (100 mg/mL) were prepared using three different mixing methods (mortar, mixer or QuartetRx) from either bulk powder, capsule content, or whole capsules. Two brands of capsules were tested in this study. All formulations were stored at room temperature (25°C / 60% RH) in amber plastic bottles for 90 days and amber plastic syringes for 14 days. Physical stability was assessed visually, while chemical stability was evaluated using a stability-indicating high-performance liquid chromatography method. Chemical derivatization with xanthydrol allowed the retention of hydroxyurea on a reverse-phase column. At least 93.9% and 97.0% of the initial concentration of hydroxyurea remained after 90 days in bottles and 14 days in syringes, respectively. There were no visual changes in formulations over the study period. Changes in pH up to 1.6 units were observed after 90 days of storage and were explained most likely by an ammonium generating degradation pathway. Ammonium was quantified and remained within safe levels in each HU 100 mg/mL oral preparations. Hydroxyurea oral liquids were all stable for 90 days in amber plastic bottles and 14 days in amber plastic syringes.

Treatment with Hydroxyurea Leads to Fetal Hemoglobin Reactivation through CA1 and LIN28B Genes: An In Vitro Study

Hydroxyurea (HU) is an effective drug to increase fetal γ-globin gene (Hb F) expression, replacing the missing adult β-globin gene. The mechanism of Hb F induction by HU and improvement in clinical symptoms are still poorly understood. The current study aimed to improve the molecular understanding of drug-induced alterations and reveals genes related to HU treatment responsiveness in β-thalassemia (β-thal). We analyzed the GSE109186 dataset using system biology and weighted gene coexpression network analysis (WGCNA) to identify and quantify gene expression changes reflected in the HU-treated human erythroblastic leukemia cells. The K562 cell line was treated in 50, 100, and 150 µM concentrations of HU for 24, 48, and 72 hours with three replications. The alteration of CA1, LIN28B and Hb F gene expression in HU-treated cells was evaluated using the real-time polymerase chain (real-time PCR) technique. The results showed that LIN28B has an increase of 4.27-fold on the first day of HU-treatment in 50 µM (p < 0.01). The CA1 expression showed a decrease at all times and doses of treatment, and the most decrease happened in 48 hours and 50 µM (p < 0.04). Hb F also showed the highest increase in 100 µM after 24 hours of treatment (5.18-fold). In summary, the data suggest that alteration of LIN28B and CA1 gene expression is associated with γ-globin increasing in HU-treated cells.

Investigating the Potential Use of Andrographolide as a Coadjuvant in Sickle Cell Anemia Therapy

Andrographolide is one of the main active principles of Andrographolide paniculata and has been extensively explored for its therapeutic use. Current studies focus on phytotherapeutics-based adjuvant therapy to symptomatically treat sickle cell anemia (SCA) as there is no specific drug/gene therapy available to date. The present study aimed to explore the potential of andrographolide as an adjuvant therapy for SCA in the presence or absence of hydroxyurea (HU), a key drug for SCA treatment. A panel of ex vivo and in vivo experimentations was performed to explore the antisickling activity of andrographolide, followed by evaluating pharmacokinetic and pharmacodynamic (PK/PD) activities in the presence of HU. Andrographolide showed significant antisickling activity using blood from SCA patients (ex vivo) and did not show any deleterious effect to cause hemolysis using rat blood (ex vivo). It displayed a substantial decrease in HU-induced decline in splenic lymphocyte proliferation and cytokine level (TNF-α and IFN-γ) using rat splenocytes (ex vivo). Concomitant oral administration of andrographolide with HU in rats for 15 days exhibited a noticeable improvement in the RBC count and hemoglobin levels comparable to the efficacy of l-glutamine (in vivo). Simultaneous administration of andrographolide with HU caused no marked effect on any pharmacokinetic parameters of HU except the highest plasma concentration of HU and its corresponding time point, which significantly dropped and delayed, respectively (in vivo). No considerable effect of andrographolide was observed on urease and horseradish peroxidase activity (in vitro). Overall, results suggest that andrographolide has several beneficial actions to be an adjuvant therapy to symptomatically manage SCA, but it should be avoided during the prescribed therapy of HU.

Biochemical Evaluation of the Effects of Hydroxyurea in Vitro on Red Blood Cells.

Hydroxyurea (HU) is a low-cost, low-toxicity drug that is often used in diseases, such as sickle cell anemia and different types of cancer. Its effects on the red blood cells (RBC) are still not fully understood. The in vitro effects of HU were evaluated on the biochemical parameters of the RBC from healthy individuals that were treated with 0.6 mM or 0.8 mM HU for 30 min and 1 h. After 30 min, there was a significant increase in almost all of the parameters analyzed in the two concentrations of HU, except for the pyruvate kinase (PK) activity. A treatment with 0.8 mM HU for 1 h resulted in a reduction of the levels of lipid peroxidation, Fe3+, and in the activities of some of the enzymes, such as glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), and PK. After the incubation for 1 h, the levels of H2O2, lipid peroxidation, reduced glutathione (GSH), enzymatic activity (hexokinase, G6PD, and superoxide dismutase (SOD) were reduced with the treatment of 0.8 mM HU when compared with 0.6 mM. The results have suggested that a treatment with HU at a concentration of 0.8 mM seemed to be more efficient in protecting against the free radicals, as well as in treating diseases, such as sickle cell anemia. HU appears to preferentially stimulate the pentose pathway over the glycolytic pathway. Although this study was carried out with the RBC from healthy individuals, the changes described in this study may help to elucidate the mechanisms of action of HU when administered for therapeutic purposes.

Pharmacokinetics of Hydroxyurea Therapy in African Children with Sickle Cell Anemia:Â Noharm Ancillary PK Study

▪Background. Hydroxyurea is an important disease-modifying therapy for sickle cell anemia (SCA). Through induction of fetal hemoglobin and other mechanisms, hydroxyurea reduces the number and severity of vaso-occlusive painful crises and episodes of acute chest syndrome, while also lowering the frequency of transfusions and hospitalizations, and even reducing mortality. In the US, hydroxyurea is recommended widely for both children and adults with SCA, although variation in treatment response is well-recognized. In sub-Saharan Africa, which has the greatest global burden of SCA, hydroxyurea is not used widely, due in part to a lack of data regarding its safety and efficacy. Pharmacokinetics (PK) analysis of hydroxyurea has also not been performed to date in Africa, but would provide important data regarding the variable responses to hydroxyurea treatment in low-resource settings.Objectives. There were three main goals of the NOHARM Ancillary PK Study: (1) to evaluate the feasibility of hydroxyurea sparse sampling PK collection and analysis in a low-resource clinical setting; (2) to determine the PK parameters for a cohort of young African children with SCA receiving hydroxyurea at an average oral daily dose of 20 mg/kg; and (3) to compare the PK parameters of African children collected and analyzed in the NOHARM trial with those of US children with SCA from the earlier HUSTLE study that included African-American children with SCA.Methods. NOHARM (NCT01976416) is a prospective, randomized, placebo-controlled, double-blinded phase 3 trial that is investigating the effects of hydroxyurea on the incidence and severity of malaria in very young children living in Uganda with SCA. After a year of blinded treatment, all participants were offered open-label hydroxyurea at an average oral daily dose of 20 ± 2.5 mg/kg, using either a 5-day or 7-day per week treatment regimen. After written informed consent for the ancillary PK study, hydroxyurea analysis was performed using a published sparse-sampling technique based on Bayesian estimation with collections pre-dose and then at T=15 minutes, 60 minutes, and 180 minutes. These four timed finger-prick sample collections over 3 hours used a volumetric absorptive microsampling device that collects exact 10 uL aliquots. All samples were stored at -80°C until analysis of hydroxyurea by LC-MS/MS (ug/mL) using a stable isotope internal standard, performed at Cincinnati Children's Hospital. Population PK analysis was performed using nonlinear mixed effect modeling (NONMEM 7.2). Allometric scaling was used to correct for differences in size.Results. A total of 100 children were enrolled in the NOHARM ancillary PK study (average age 3.8 ± 0.9 years; 55 males), and 98 of these children had three evaluable post-dose samples. Individual PK profiles were best described by a one-compartment model with first order absorption. The dose-normalized hydroxyurea concentrations in NOHARM were comparable between the 5-day and 7-day dosing regimens and similar to published HUSTLE data for 96 children, except for higher T=180 hydroxyurea concentrations and overall higher hydroxyurea exposure in the NOHARM cohort. The higher blood hydroxyurea concentrations were the result of reduced drug clearance in NOHARM, possibly influenced by younger age. Population PK parameter estimates of oral clearance in the NOHARM and HUSTLE cohorts were 9.8 L/h/70kg versus 12.9 L/h/70kg, respectively (p<0.01).Conclusions. The NOHARM PK study demonstrates the feasibility of collecting timed samples for analysis of hydroxyurea exposure, even in the low-resource setting of Uganda. Using a sparse sampling technique, young children with SCA had individual PK parameters calculated and a population PK model established. The oral drug clearance was lower in NOHARM than HUSTLE, with resulting higher hydroxyurea exposure, perhaps reflecting differences in age, nutrition, glomerular filtration, and genetic modifiers that warrant further investigation. DisclosuresWare:Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Personalized Hydroxyurea Dosing to Reduce Time to MTD and Optimize the HbF Response: Results from the TREAT Study

▪Background: Hydroxyurea is well established as the standard of care for children with sickle cell anemia (SCA) in the US, but current dosing schemes and dose responses for individual patients are highly variable. Traditionally, hydroxyurea is started at a low dose of 15-20 mg/kg/day, then slowly escalated to maximum tolerated dose (MTD) based upon hematological parameters to avoid toxicities. Using this approach, the average hydroxyurea MTD is about 25 mg/kg/day and yields an average HbF induction of 20-25%, but the time to achieve MTD is more than 6 months. We developed and prospectively evaluated a population pharmacokinetics (PK)-based dosing model of hydroxyurea for children with SCA, aiming to initiate hydroxyurea at a personalized dose that achieves MTD quickly and achieves a more robust HbF response.Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154) is a prospective sudy with a primary objective to develop and evaluate a population PK-based model to predict the hydroxyurea MTD through an individualized dosing strategy. The study recruited patients (age 6 months to 18 years) who were initiating hydroxyurea therapy. Prior to starting hydroxyurea, each participant took a single oral dose of 20 mg/kg with subsequent post-dose sampling at 15, 60, and 180 minutes. This sparse sampling strategy was based upon our previously described population PK model that allows accurate estimation of hydroxyurea exposure using only three post-dose blood samples. Plasma hydroxyurea concentrations were measured by HPLC. Published PK data from children who had reached MTD demonstrated that the mean hydroxyurea exposure (AUC) at MTD was 115 mg×h/L. TREAT incorporated individual PK data for each participant using population PK model based Bayesian estimation, to generate a starting hydroxyurea dose that would target this AUC goal in an attempt to start at a dose near or at MTD. Clinical follow-up and dose adjustments were subsequently provider-dependent and based upon institutional clinical guidelines recommending dose escalation to MTD, based on a target ANC of less than 3.0 x10^6/L. The official determination of MTD was declared by two independent hematologists blinded to patient identity; MTD was defined as a stable daily dose without dose escalation, holds, or decreases for at least 8 weeks, along with laboratory values within the target range. The primary outcome of TREAT was to reduce the time to MTD by at least two months from an average of published studies demonstrating a mean (±SD) of 9±4 months.Results:A total of 38 children enrolled in TREAT had PK studies performed to generate an individualized starting dose. Only three participants (7.9%) had incomplete PK studies that did not allow for an individualized starting dose and were started at 20 mg/kg. The enrolled children were young with an average (mean±SD) age of 3.1±4.3 years. Most participants (26/38=68%) were less than two years of age at enrollment, while half (19/38=50%) were less than one year of age. For participants starting with PK-based dosing (n=35), the average starting hydroxyurea dose was 27.5±4.5 mg/kg/day, with 12/35 children (34%) starting hydroxyurea at doses ≥30 mg/kg/day. Despite the high starting doses, there were very few hematological toxicities, with only 2/35 participants requiring a dose decrease from the starting dose due to moderate neutropenia. The individualized starting dose was the actual MTD for 73% of participants without the need for a single dose escalation. The mean time to MTD was 6.2 months (median 4 months) compared to a historical baseline of 9 months. Even with high starting HbF (24.9±11.6%), response was excellent with all participants who have completed at least 6 months of hydroxyurea demonstrating an increase in HbF from baseline. The mean maximum achieved HbF is 35.5±9.5% with 74% achieving HbF>30% and 35% achieving HbF>40%.Conclusions: TREAT has demonstrated the feasibility of individualized, PK-guided hydroxyurea therapy and suggests that early initiation of hydroxyurea for young children with SCA is both safe and effective, achieving HbF responses (>30-40%) beyond which has been seen with standard dosing. This precision medicine approach to hydroxyurea therapy suggests that it is possible to exceed the traditional therapeutic goals with individualized dosing and could be transformational for the treatment of young children with SCA. DisclosuresQuinn:Global Blood Therapeutics: Research Funding. Ware:Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Effect of Concomitant Hydroxyurea Therapy with Rutin and Gallic Acid: Integration of Pharmacokinetic and Pharmacodynamic Approaches.

Hydroxyurea (HU) is the first-ever approved drug by USFDA for sickle cell anemia (SCA). However, its treatment is associated with severe side effects like myelosuppression. Current studies are focused on the supplementation therapy for symptomatic management of SCA. In the present study, we aimed to explore rutin’s and gallic acid’s potential individually, for concomitant therapy with HU using pharmacokinetic and pharmacodynamic approaches since there is no such precedent till date. In vivo pharmacokinetic studies of HU in rats showed that rutin could be safely co-administered with HU, while gallic acid significantly raised the plasma concentration of HU. Both the phytochemicals did not have any marked inhibitory effect on urease but have considerable effects on horseradish peroxidase enzyme. The experimental phytoconstituents displayed a very low propensity to cause in vitro hemolysis. Gallic acid markedly enhanced the HU-induced decrease in lymphocyte proliferation. A substantial improvement by rutin or gallic acid was observed in HU-induced reduction of the main hematological parameters in rats. Combined treatment of HU with rutin and gallic acid reduced serum levels of both IL-6 and IL-17A. Overall, both rutin and gallic acid are found to have promising phytotherapy potential with HU. Further exploration needs to be done on both candidates for use as phytotherapeutics for SCA.

Role of β 3 ‐Adrenergic Receptors in Rat Aortic Endothelial Cell Function

Long-term success of percutaneous trans-luminal coronary angioplasty (PTCA) is limited by the frequent occurrence of restenosis, resulted from vascular smooth muscle cell (VSMC) proliferation and migration due to the procedure-induced endothelial damage. Speeding the healing of the endothelial layer has been considered as one of the major approaches to reduce the risk of restenosis. Basic fibroblast growth factor (bFGF), a major growth factor released after injury, regulates endothelial cell proliferation and migration and promotes endothelial layer healing. Our previous results revealed that β3-adrenergic receptor (β3-ARs) expression is upregulated in newly formed intima in injured blood vessels and activation of β3-ARs enhanced bFGF-induced VSMC proliferation and migration. However, the role of β3-ARs in endothelial function, specifically in injured arteries, is uncertain. The purpose of this study is to determine if activation of endothelial cell β3-ARs potentiates bFGF-induced endothelial cell proliferation and/or migration by using rat aortic endothelial cells. Cell proliferation was determined by measuring viable cell numbers using Resazurin. Cell migration will be quantified by a monolayer wound healing assay in the presence of a cell proliferation inhibitor, hydroxyurea. A dose-dependent inhibition of cell proliferation was performed to optimize the concentration of hydroxyurea in cell migration experiments. Our results revealed that pretreatment of endothelial cells with a selective β3-AR agonist, CL316,243, enhanced bFGF-induced vascular endothelial cell proliferation. We also found that 5μM of hydroxyurea completely inhibited cell proliferation induced by growth factors. In conclusion, our data suggest that activation of β3-ARs may speed the healing of endothelial layer by promoting cell proliferation and that including 5μM of hydroxyurea in cell migration experiments will allow us to measure the cell motility accurately and ensure that proliferation does not take place.

Hydroxyurea selection for enhancement of homology-directed targeted integration of transgenes in CHO cells

Site-specific integration via genome editing technologies has been implemented in Chinese hamster ovary (CHO) cells for predictable and efficient cell line development and engineering. Various strategies have been employed to enhance knock-in (KI) efficiency for precise homology-directed repair (HDR)-mediated targeted integration of transgenes in CHO cells. Given the cell cycle-dependent regulation of the DNA damage repair pathway, cell cycle synchronization to the HDR-favored S/G2 phase has been successfully utilized in mammalian cells, but the effect is limited in CHO cells. Here, we describe a cell cycle enrichment method to increase HDR-mediated KI efficiency in CHO cells. Existing G1 cell cycle synchronization methods showed transient cell cycle arrest and did not improve KI efficiency. Rather than cell cycle arrest with a high concentration of chemicals followed by a release step, cells were incubated in the presence of a lower concentration of hydroxyurea (HU) to enrich cells in the S phase. HU selection allowed for robust S phase enrichment of CHO cells by up to 70 % and maintained cell viability. This short-term selection resulted in improved KI efficiency by 1.2–1.5 fold compared with cells in the control condition. Overall, this approach serves as a simple and effective strategy for enhancement of site-specific genome engineering in CHO cells.

Electrochemical Determination of Hydroxyurea in a Complex Biological Matrix Using MoS2-Modified Electrodes and Chemometrics.

Hydroxyurea, an oral medication with important clinical benefits in the treatment of sickle cell anemia, can be accurately determined in plasma with a transition metal dichalcogenide-based electrochemical sensor. We used a two-dimensional molybdenum sulfide material (MoS2) selectively electrodeposited on a polycrystalline gold electrode via tailored waveform polarization in the gold electrical double layer formation region. The electro-activity of the modified electrode depends on the electrical waveform parameters used to electro-deposit MoS2. The concomitant oxidation of the MoS2 material during its electrodeposition allows for the tuning of the sensor’s specificity. Chemometrics, utilizing mathematical procedures such as principal component analysis and multivariable partial least square regression, were used to process the electrochemical data generated at the bare and the modified electrodes, thus allowing the hydroxyurea concentrations to be predicted in human plasma. A limit-of-detection of 22 nM and a sensitivity of 37 nA cm−2 µM−1 were found to be suitable for pharmaceutical and clinical applications.

Endogenous p21-Dependent Transgene Control for CHO Cell Engineering.

Numerous engineering efforts have been made in Chinese hamster ovary (CHO) cells for high level production of therapeutic proteins. However, the dynamic regulation of transgene expression is limited in current systems. Here, we investigated the effective regulation of transgene expression in CHO cells via targeted integration-based endogenous gene tagging with engineering target genes. Targeted integration of EGFP-human Bcl-2 into the p21 locus effectively reduced the apoptosis, compared with random populations in which Bcl-2 expression was driven by cytomegalovirus (CMV) promoter. Endogenous p21 and EGFP-human Bcl-2 displayed similar expression dynamics in batch cultures, and the antiapoptotic effect altered the expression pattern of endogenous p21 showing the mutual influences between expression of p21 and Bcl-2. We further demonstrated the inducible transgene expression by adding low concentrations of hydroxyurea. The present engineering strategy will provide a valuable CHO cell engineering tool that can be used to control dynamic transgene expression in accordance with cellular states.

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.