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.

Eritrocitose primária em cão: relato de caso / Primary erythrocytosis in dog: case report

A eritrocitose absoluta primária, também denominada de policitemia vera, é um distúrbio mieloproliferativo crônico de causa desconhecida, caracterizado pela proliferação clonal de células-tronco eritróides neoplásicas. Acomete cães de meia-idade entre seis e sete anos. As manifestações clínicas mais comuns são letargia, fraqueza, poliúria, polidipsia, sangramentos como epistaxe, hematúria, hematoemese, hematoquezia, até mesmo convulsões e ataxia. O diagnóstico é baseado em valores altos de hematócrito, geralmente acima de 70%, excluindo-se as causas de eritrocitose secundária. As concentrações séricas de eritropoietina estão normais ou diminuídas. O tratamento consiste em flebotomia e administração de hidroxiuréia. Relata-se o caso de uma cadela, raça Bichon Frise, 11 anos, que, no início do quadro, apresentou hematócrito de 84%, letargia, ataxia, mucosas congestas, cianose de língua, poliúria e polidipsia. Realizou-se o tratamento com hidroxiuréia durante oito anos, na dose de 15 a 30 mg/kg, a cada 24 horas, sem ocorrência de efeitos colaterais ou recidiva das manifestações clínicas.(AU)

Primary absolute erythrocytosis, also termed polycythemia vera, is a chronic myeloproliferative disorder of unknown cause. It is characterized by clonal proliferation of neoplastic erythroid stem cells. It affects middle-aged dogs between 6-7 years. The most common clinical manifestations are lethargy, weakness, polyuria, polydipsia, and bleeding such as epistaxis, hematuria, hematoemese, and hematochezia. Seizures and ataxia are also common. Diagnosis is based on high hematocrit values, generally above 70% excluding the causes of secondary erythrocytosis. Serum concentrations of erythropoietin are at a normal level or decreased level. Treatments consists of hydroxyurea and phlebotomy management. It is reported that case of female Bichon Frise, 11 years old who onset of the disease had a hematocrit of 84%, lethargy, ataxia, congested mucous membranes, tongue cyanosis, polyuria and polydipsia. The treatment with hydroxyurea was performed for 8 years, at a dose of 15 to 20mg/kg, every 24 hours, without occurrence of side effects or recurrence of clinical manifestations.(AU)

Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

Hydroxyurea (HU) is the most important advance in the treatment of sickle cell anaemia (SCA) for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH) and Methyl ThiazolTetrazolium (MTT) and the comet assay to investigate the cytotoxicity and damage index (DI) of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years), and the group of healthy individuals (AA) used as a control group (n=52, 28 women and 24 men, aged 19-60 years), The SSHU group (n=21, 11 women and 10 men, aged 19-63 years) showed a significant reduction (p<0.001) in LDH activity and an increase in the percentage of viable cells by the MTT (p<0.001). However, the SSHU group presented significantly higher DI values (49.57±6.0 U/A) when compared to the AA group (7.43 ± 0,94U/A) and the SS group (22.73 ±5.58 U/A) (p<0.0001), especially when treated for longer periods (>20 months), demonstrating that despite the cytoprotective effects in terms of cell viability, the use of HU can induce DNA damage in neutrophils.

A hidroxiuréia (HU) constitui o avanço mais importante no tratamento da anemia falciforme (AF) por prevenir complicações e aumentar a qualidade de vida dos pacientes. Entretanto, alguns aspectos do tratamento com HU permanecem obscuros, incluindo a sua ação e potencial toxicidade em outras células sanguíneas, tais como neutrófilos. Este estudo utilizou a mensuração da lactato desidrogenase (LDH) e do metil tiazoltetrazólio (MTT) e o ensaio do cometa para investigar a citotoxicidade e índice de dano (ID) ao DNA em neutrófilos de pacientes com AF em uso do medicamento. Nos ensaios de LDH e MTT, observou-se além de ausência de toxicidade, uma ação citoprotetora no grupo de pacientes tratados, Grupo SSHU (n=21, 11 mulheres e 10 homens, com idades entre 19-63 anos), quando comparados aos pacientes sem tratamento, Grupo SS (n=20, 13 mulheres e 07 homens, 18-69 anos), e grupo de indivíduos saudáveis (AA) usado como controle (n=52, 28 mulheres e 24 homens, 19-60 anos), com redução significativa (p<0,001) na atividade de LDH e aumento no percentual de células viáveis pelo MTT (p<0,001). Entretanto, o grupo SSHU apresentou valores de ID significativamente elevados (49,57±6,0 U/A), quando comparados ao grupo AA (7,43 ± 0,94U/A) e grupo SS (22,73 ±5,58 U/A) (p<0,0001), especialmente quando tratados por períodos mais longos (>20 meses), demonstrando que apesar dos efeitos citoprotetores quanto à viabilidade celular, o uso da HU pode induzir lesão ao DNA de neutrófilos.

Standardization method for measurement of hydroxyurea by Ultra High Efficiency Liquid Chromatography in plasma of patients with sickle cell disease

Sickle cell anemia (SCA) is a recessively inherited disease characterized by chronic hemolytic anemia, chronic inflammation, and acute episodes of hemolysis. Hydroxyurea (HU) is widely used to increase the levels of fetal hemoglobin (HbF). The objective of this study was to standardize and validate a method for the quantification of HU in human plasma by using ultra high performance liquid chromatography (UPLC) in order to determine the plasma HU levels in adult patients with SCA who had been treated with HU. We used an analytical reverse phase column (Nucleosil C18) with a mobile phase consisting of acetonitrile/water (16.7/83.3). The retention times of HU, urea, and methylurea were 6.7, 7.7, and 11.4 min, respectively. All parameters of the validation process were defined. To determine the precision and accuracy of quality controls, HU in plasma was used at concentrations of 100, 740, and 1600 µM, with methylurea as the internal standard. Linearity was assessed in the range of 50-1600 µM HU in plasma, obtaining a correlation coefficient of 0.99. The method was accurate and precise and can be used for the quantitative determination of HU for therapeutic monitoring of patients with SCA treated with HU.

A anemia falciforme (AF) é uma doença hereditária recessiva caracterizada por anemia hemolítica crônica, inflamação crônica e episódios agudos de hemólise. Hidroxiureia (HU) é amplamente utilizada para aumentar os níveis de hemoglobina fetal (Hb F). O objetivo consiste em padronizar e validar um método para a quantificação de HU no plasma humano utilizando Cromatografia Líquida de Ultra Alta Eficiência (UPLC), a fim de determinar os níveis de HU em pacientes adultos com AF, tratados com HU. Utilizou-se coluna analítica de fase inversa (Nucleosil C18), fase móvel constituída por acetonitrila/água (16,7/83,3). Os tempos de retenção da HU, uréia e metiluréia foram respectivamente de 6,7, 7,7 e 11,4 minutos. Definiram-se todos os parâmetros do processo de validação. Para determinar a precisão e exatidão dos controles de qualidade utilizaram-se concentrações de 100, 740 e 1600 mM de HU no plasma, empregando como padrão interno a metiluréia. A linearidade foi avaliada no intervalo de 50 1600 mM de HU no plasma, obtendo-se coeficiente de correlação de 0,99. O método foi considerado exato e preciso e pode ser realizado com o propósito de determinação quantitativa de HU para monitorização terapêutica de pacientes com AF tratados com esse fármaco.

Chemical and functional analysis of generic hydroxyurea formulations.

Hydroxyurea has documented laboratory and clinical efficacy for children with sickle cell anemia (SCA), and has potential to become an effective and inexpensive treatment option for patients in countries with limited resources. Concerns exist, however, regarding product quality and manufacturing variability among different international vendors, particularly for generic formulations. To address these concerns, hydroxyurea capsules from 8 different pharmaceutical sources were analyzed using quantitative chemical and functional assays. All samples had measured values within 20% of expected results, with no significant differences observed among vendors. Generic hydroxyurea formulations represent a potent yet inexpensive therapeutic option for children with SCA worldwide.

Plasma and urine hydroxyurea levels might be useful in the management of adult sickle cell disease.

Hydroxyurea (HU) is useful for treating sickle cell anemia because of its ability to reduce some of the severe clinical events such as painful crises and acute chest syndrome. It may also reduce the need for blood transfusions and frequent hospitalizations and reduce mortality. Nevertheless, no consistent recommendations regarding its therapeutic schedule are defined. Our aim was to improve and validate a high performance liquid chromatography (HPLC) technique to measure HU and to study HU levels in serum and urine of sickle cell anemia patients and relate this to treatment efficacy and compliance. Thirty-seven patients received 1,128 +/- 333 mg of HU per day (8.0 to 28.0 mg/kg/day). Plasma and/or urine were sampled and HU was measured using an HPLC method coupled with UV detection. We validated a specific, sensitive assay with good reproducibility and linearity, and showed a positive relationship between plasma HU concentrations and time elapsed between oral HU intake and sampling. We observed plasma HU concentrations were positively correlated with change in mean corpuscular volume (MCV) before and during the treatment. No correlation was obtained between HU concentration and Hb F level.

Determination of hydroxyurea in capsules and biological fluids by ion-selective potentiometry and fluorimetry.

Two hydroxyurea selective electrodes were investigated with beta-cyclodextrin used as ionophore and either tetrakis (p-chlorophenyl) borate (electrode 1), or tetrakis [3,4-bis (trifluoromethyl) phenyl] borate (electrode 2), as a fixed anionic site in a polymeric matrix of carboxylated polyvinyl chloride. Linear responses of hydroxyurea within a concentration range of 10(-5)-10(-)3 M with slopes of 51.2 and 58.6 mV/decade with pH 3-6 were obtained by using electrodes 1 and 2, respectively. Two spectrofluorimetric methods involving the formation of drug-AI(III) complex (method 3) and drug-Mg(II) complex (method 4) at pH 5 were also investigated. These complexes emit fluorescence at wavelengths of 380 and 355 nm, after excitation at 305 nm, for AI and Mg complexes, respectively. The calibration graphs were rectilinear from 0.5 to 2.5 microg/mL for the AI complex and 1 to 5 microg/mL for the Mg complex. The 4 proposed methods display useful analytical characteristics for determination of hydroxyurea, with average recoveries of 100.2 +/- 0.83 and 99.4 +/- 1.81% in capsules and 99.7 +/- 0.70 and 99.4 +/- 1.25% in biological fluids for the potentiometric and fluorimetric methods, respectively. Results obtained by the proposed procedures were statistically analyzed and compared with those obtained by the U.S. Pharmacopeial method. The 4 proposed procedures were also used to determine the stability of the drug in the presence of its degradate, hydroxylamine.

Determination of hydroxyurea in plasma and peritoneal fluid by high-performance liquid chromatography using electrochemical detection.

A sensitive method has been developed for the determination of hydroxyurea in plasma and peritoneal fluid using reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Plasma or peritoneal fluid samples were treated with acetonitrile to precipitate proteins then injected to the HPLC. A C18 analytical column was used to separate hydroxyurea from interfering substances in the biological matrix. The mobile phase, consisting of 0.2 M sodium perchlorate-methanol (95:5, v/v) adjusted to pH 5.0, was delivered isocratically at a flow-rate of 1 ml/min and hydroxyurea was detected using a glassy-carbon electrode operating at an applied potential of +800 mV. Hydroxyurea eluted with a retention time of 3 min. The cycle time for analysis is short and the assay precision is acceptable (C.V. plasma=1.4-3.9%. C.V. peritoneal fluid=2.1-9.7%). The method has been validated and is linear from 25 to 400 ng/ml in plasma and 5 to 30 ng/ml in peritoneal fluid. The method has been shown to be applicable for pharmacokinetic studies.

Determination of 1-benzo[b]thien-2-ylethanone and related impurities by high performance liquid chromatography.

1-Benzo[b]thien-2-ylethanone (2-acetylbenzothiophene, 2-ABT) and related impurities were determined using a reverse-phase high performance liquid chromatography system and UV detection at 254 nm. Separation was achieved isocratically on a 4.6 mm x 25 cm, 5 microns Zorbax Rx-C8 column using an eluent which is 0.2% perchloric acid/THF in a ratio of 60:40 (v/v). The chromatographic system resolved 2-ABT and known impurities in less than 45 min with near baseline resolution. Known impurities were quantitated versus 2-ABT with corrections made for differences in detector response at the specified wavelength. Linearity for 2-ABT was demonstrated with a correlation coefficient > 0.9999. Assay precision (RSD values) for impurities at 0.5% ranged from +/- 1.8% to +/- 14%, while precision (RSD values) for the 2-ABT determination ranged from +/- 0.81% to +/- 1.1%. A variety of different chromatographic columns and conditions are discussed for the application.

Analysis of hydroxyurea in capsules and aqueous solution and stability study with capillary gas chromatography and thermionic (N-P) specific detection.

A method for the analysis of hydroxyurea (HU) in solutions, powder, or capsules by use of capillary gas chromatography with N-P thermionic specific detection is described. Upon injection of an HU solution in a methanol and acetone mixture, the drug formed pyridine which was well separated from the internal standard (thiotepa) on a 30-m fused-silica, SE-30 capillary column with temperature programming. The peak height ratio versus concentration standard curves were linear with correlation coefficient ranging between 0.9942 and 0.9993. The coefficients of variation at 5, 25, and 50 micrograms/L were 7.2, 5.7, and 5.5%, respectively. Hydroxyurea was extracted from powder or capsule formulations with a mixture of methanol and acetone (50:50, v:v), and the percentage found of the label claim for 10 capsules ranged between 96.7 and 104.9 (mean = 100.1; CV = 2.7%). Further, this assay was used to examine the stability of hydroxyurea in aqueous solutions at 4, 23, and 45 degrees C, and the apparent first-order rate constants observed at these temperatures were 0.06407, 0.08113, and 0.1293 day-1, respectively; the activation energy was 3011 cal.K-1.mol-1.

Quantitative analysis of hydroxyurea and urea by proton nuclear magnetic resonance (NMR) spectroscopy.

Quantitative 1H-nuclear magnetic resonance (NMR) procedures are described for measuring hydroxyurea and urea. Dimethylsulfoxide-d6 is used as the solvent for both assays; the internal standards employed are urea and p-dichlorobenzene for hydroxyurea and urea, respectively. The analysis for hydroxyurea is based on the comparison of the area of the -NH2 peak of hydroxyurea with the area of the urea -NH2 peak area. The analysis of urea is based on the comparison of the -NH2 peak of urea with the area of the p-dichlorobenzene singlet. The 1H-NMR assays yield results that are precise and agree well with results of the more cumbersome and less specific USP procedures.

A rapid colorimetric assay for carbamyl phosphate synthetase I.

A rapid, reproducible, and sensitive colorimetric assay for carbamyl phosphate synthetase I was presented. A four-fold increase in sensitivity and reduced assay time were afforded by this procedure. The method utilized the chemical conversion of carbamyl phosphate to hydroxyurea by the action of hydroxylamine instead of employing a coupling enzyme. The hydroxyurea was quantitated in 15 min by an improved colorimetric assay for ureido compounds by measuring the absorption of the resulting chromophore at 458 nm. Optimum conditions for both the formation and quantitation of hydroxyurea were established. Activity measurements of carbamyl phosphate synthetase I obtained by this uncoupled method were identical with those obtained by the ornithine transcarbamylase coupled assay.

Solvent-dependent conformational system in hydroxyureas detected by NMR spectroscopy.

The proton magnetic resonance spectra of the antileukemia agent hydroxyurea and substituted hydroxyureas in several solvents were recorded and correlated with structural features. Solvent-dependent differences in conformational preferences due to effects on internal hydrogen bonding, a temperature-dependent conformational feature, anthe exchangeability of protons with deuterium oxide and acetone-d6 were observed. The conformational features consistent with the spectral data are discussed.