Chelators as Antineuroblastomas Agents.

Neuroblastoma represents 8-10 % of all malignant tumors in childhood and is responsible for 15 % of cancer deaths in the pediatric population. Aggressive neuroblastomas are often resistant to chemotherapy. Canonically, neuroblastomas can be classified according to the MYCN (N-myc proto-oncogene protein) gene amplification, a common marker of tumor aggressiveness and poor prognosis. It has been found that certain compounds with chelating properties may show anticancer activity, but there is little evidence for the effect of chelators on neuroblastoma. The effect of new chelators characterized by the same functional group, designated as HLZ (1-hydrazino phthalazine), on proliferation (WST-1 and methylene blue assay), cell cycle (flow cytometry), apoptosis (proliferation assay after use of specific pharmacological inhibitors and western blot analysis) and ROS production (fluorometric assay based on dichlorofluorescein diacetate metabolism) was studied in three neuroblastoma cell lines with different levels of MYCN amplification. The molecules were effective only on MYCN-non-amplified cells in which they arrested the cell cycle in the G0/G1 phase. We investigated the mechanism of action and identified the activation of cell signaling that involves protein kinase C.

Murine experimental models for studying the pathogenesis of coxsackieviruses.

Understanding the pathogenesis of communicable diseases often involves animal models. Mouse models are studied by researchers to achieve a better understanding of the relationship between the biological, physiochemical, and antigenic properties of the infectious agent, as well as the histopathological, immunological, and functional changes in the living system and the target organs, in short, the pathophysiological processes that the communicable agents bring about. The long-term objectives of the in vivo studies are important from a medical point of view, as they represent faithful (reliable and similar) human responses, which enhance the development of diagnostics, treatment, and measures for preventing the spread of the disease. Our work is devoted to the murine models used for understanding the pathogenesis of coxsackieviruses. We describe different mouse models used for studying the diseases caused by coxsackieviruses and the immune responses in different mouse models.  We then shortly elucidate experiences from our laboratory related to the oral route of infection, and compare the similarities and differences we found in this model. Keywords: pathogenesis; coxsackievirus; murine models.

Small molecule inhibitors of DNA-PK for tumor sensitization to anticancer therapy.

The most sensitive cell structure - a DNA molecule, is the common target of cancer therapy. DNA damage response (controlled by enzymes from the phosphatidylinositol 3-kinase-related kinases family - PIKK) presents many encouraging targets for improving both conventional cytotoxic anticancer therapy and individualized monotherapy. DNA-dependent protein kinase (DNA-PK) is a member of the PIKK superfamily and plays an important role in the detection and repair of DNA double-strand breaks via the non-homologous end-joining pathway. The ability of cancer cells to repair DNA damage is an important element determining their sensitivity to radio- or chemo-therapy. The overactivation of DNA-PK in cancers can result in resistance to anticancer therapy. The inhibition of DNA-PK is a very promising target in anticancer research. However, the specific DNA-PK inhibitors currently known are limited by poor solubility and high metabolic lability in vivo, leading to a short serum half-life. Construction of new compounds based on existing drugs is the most important strategy to improve drug efficacy, pharmacokinetic parameters and to reduce toxicity. This review will describe small molecule inhibitors and summarize their efficacy in synergizing radio- and chemotherapy in vitro.

Fiber-optic pH detection in small volumes of biosamples.

Determining the pH values of microscopic plant samples may help to explain complex processes in plants, so it is an area of interest to botanists. Fiber-optic probes with small dimensions can be used for this purpose. This paper deals with the fiber-optic detection of the pH values of droplets of plant xylem exudate based on ratiometric fluorescence intensity measurements with an internal reference. For this purpose, novel V-taper sensing probes with a minimum diameter of around 8 µm were prepared that enable the delivery of fluorescence signal from the detection site on the taper tip to the detector. The taper tips were coated with pH-sensitive transducer (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt; HPTS) and a reference [dichlorotris-(1,10-phenanthroline) ruthenium (II) hydrate (Ru-phen dichloride)] immobilized in a xerogel layer of propyltriethoxysilane and (3-glycidoxy)propyl trimethoxysilane. The prepared probes were sensitive to pH values mainly in the range from 6.0 to 9.0. In the pH range 6-9, the results were limited by measurement errors of about 0.2 pH units, and in the pH range 5-6 by measurement errors of about 0.5 pH units. Using the developed V-taper sensing probes, the pH values of in vivo and in vitro samples of small volumes (~6 µl) of exudate were measured. The results were validated by comparison with conventional electrochemical pH measurements.

[Pindolol determination in pharmaceuticals by the method of capillary isotachophoresis]. / Stanovení pindololu v lécivých prípripravcích metodou kapilární izotachoforézy.

Capillary izotachophoresis (ITP) with conductimetric detection was employed to determine pindolol (PI) in the pharmaceutical preparation Apo-pindol. The optimal operational electrolytic system was composed of 10 mM of potassium picolinate and 10 mM picolinic acid (leading electrolyte, LE; pH=5.1) and 10 mM of formic acid as the terminating electrolyte (TE). The time and currents mode of analysis were 50 microA (800 s), 10 microA. The total period of analysis was about 19 minutes. The method is suitable for the determination of PI in Apo-pindol tablets, RSD=1.45% (n=6). The method was validated from the viewpoint of precision, accuracy, and linearity of the ITP method. The accuracy of the method was determined by an analysis of a sample with an added standard and the recovery was 98.0%.

Determination of bopindolol in pharmaceuticals by capillary isotachophoresis.

Capillary isotachophoresis (ITP) with conductimetric detection has been used for separating and determining bopindolol (I) in commercial mass-produced pharmaceutical preparations. The optimised operational electrolyte system consisted of 5 mM potassium picolinate and 5 mM picolinic acid (leading electrolyte, LE; pH 5.37) and 10 mM formic acid as the terminating electrolyte (TE). The driving and detection currents were 50 microA (for 350 s) and 10 microA, respectively. The single analysis took about 12 min. Under such conditions the effective mobility of I was determined as 16.73 10(-9)m(2) V(-1) s(-1) (with tetraethylammonium as the mobility standard). The calibration graph relating the ITP zone length to the concentration of I was rectilinear (r=0.99990) in the range 10-100 mg l(-1). The relative standard deviation (R.S.D.) was 0.90% (n=6) when determining 50 mg l(-1) of I in pure test solution. Sample pre-treatment of the tablets involved ice-cooled extraction of I with methanol. The method was suitable for determining I in Sandonorm tablets with R.S.D. value 1.45% (n=6). According to the validation procedure based on the standard addition method the recovery was 97.3%.

Determination of ambroxol or bromhexine in pharmaceuticals by capillary isotachophoresis.

Expectorant drugs ambroxol (AX) and bromhexine (BX) were determined by capillary isotachophoresis (ITP) with conductimetric detection. The leading electrolyte (LE) was a buffer solution that contained 5 mM picolinic acid and 5 mM potassium picolinate (pH 5.2). The terminating electrolyte (TE) was 10 mM formic acid. The driving current was 80 microA (for approximately 200 s) or 50 microA (for approximately 350 s) and the detection current was 20 microA (a single analysis took about 8 min). The effective mobilities of AX and BX (evaluated with tetraethylammonium as the mobility standard) were 18.8 x 10(-9) m2 V(-1) s(-1) and 14.3 x 10(-9) m2 V(-1) s(-1) respectively. The calibration graphs relating the ITP zone length to the concentration of the analytes were rectilinear (r = 0.9993-0.9999) in the range 10 mg L(-1) (20 mg L(-1) for BX) to 200 mg l(-1) of the drug standard. The relative standard deviations (RSD) were 1.2 1.6% (n = 6) when determining 100 mg l(-1) of the analytes in pure test solutions. The method has been applied to the assay of AX or BX in seven commercial mass-produced pharmaceutical preparations. According to the validation procedure based on the standard addition technique the recoveries were 97.5-102.7% of the drug and the RSD values were 0.11-2.20% (n = 6).

[Determination of xanthinol nicotinate in drug preparations using capillary isotachophoresis]. / Stanovení obsahu xanthinoliumnikotinátu v lécivých prípravcích metodou kapilární izotachoforézy.

Cationic capillary isotachophoresis (ITP) with coductimetric detection has been used for separating and determination of xanthinol in two commercial mass-produced preparations. The optimized ITP electrolyte system consisted of 5 mM potassium picolinate + 5 mM picolinic acid (pH 5.21) as the leading electrolyte and 10 mM formic acid as the terminating electrolyte. The driving and detection currents were 50 microA (for 380 s) and 10 microA, respectively. The analysis took about 8 min. According to the validation procedure based on the reference UV spectrophotometric method, the ITP method gave practically the same results.

[Oxidative determination of hydroxyamphetamine in drops using flow injection analysis]. / Oxidacní stanovení hydroxyamfetaminu v kapkách metodou prutokové injekcní analýzy.

A flow injection-spectrophotometric determination at 540 nm of hydroxyamphetamine is reported. The procedure is based on a coloured reaction with 2-hydrazono-3-methylbenzothiazole (MBTH) and ceric ammonium nitrate (CeIV) as the oxidation reagent. Under optimised conditions, the determination of hydroxyamphetamine is achieved in a range of 0.01-0.2 mmol.l-1 with a relative standard deviation of 0.78% for 0.05 mmol.l-1 (n = 10) and a sample throughput of 75.h-1. The method was used for the determination of medicinal drug in the mass-produced dosage form of Czech origin: Pedrolon drops, Galena a.c.

Capillary isotachophoretic determination of flufenamic, mefenamic, niflumic and tolfenamic acid in pharmaceuticals.

Anionic capillary isotachophoresis (ITP) with conductimetric detection has been used for determining selected non-steroid anti-inflammatory and analgesic drugs of the phenamate group, namely tolfenamic (I), flufenamic (II), mefenamic (III) and niflumic (IV) acid. Initially the pKa values (proton lost) of I-IV were determined as 5.11, 4.91, 5.39 and 4.31, respectively, by the UV spectrophotometry in aqueous 50% (w/w) methanol. The optimised ITP electrolyte system consisted of 10 mM HCl + 20 mM imidazole (pH 7.1) as the leading electrolyte and 10 mM 5,5'-diethylbarbituric acid (pH 7.5) as the terminating electrolyte. The driving and detection currents were 100 microA (for 450 s) and 30 microA, respectively (a single analysis took about 20 min). Under such conditions the effective mobilities of I-IV varied between 23.6 and 24.6 m2 V(-1) s(-1) (evaluated with orotic acid as the mobility standard). The calibration graphs relating the ITP zone length to the concentration of the analytes were rectilinear (r = 0.9987-0.9999) in the range 10-100 mg l(-1) of the drug standard. The R.S.D.s were 0.96-1.55% (n = 6) when determining 50 mg l(-1) of the analytes in pure test solutions. The method has been applied to the assay of the phenamates in six commercial mass-produced pharmaceutical preparations (Mobilisin gel and ointment, Lysalgo capsules, Nifluril cream, Niflugel gel, and Clotam capsules). According to the validation procedure based on the standard addition technique the recoveries were 98.4-104.3% of the drug and the R.S.D. values were 1.25-3.32% (n = 6).

Separation and determination of sorbitol and xylitol in multi-component pharmaceutical formulations by capillary isotachophoresis.

Pharmaceutically important polyhydric alcohols sorbitol (SO) and xylitol (XY) are efficiently separated and determined by analytical capillary isotachophoresis (ITP) with conductometric detection. The on-column complex-formation equilibria between the polyols and boric acid are utilized--the terminating borate ion acts as the complexing agent. The ITP operational system used consists of 10 mM HCl + 20 mM imidazole (LE, pH 7.0) and 20 mM boric acid (TE, pH 8.0). The effective mobilities of the borated SO and XY are 8.3 x 10(-9) m2 V-1 s-1 and 7.4 x 10(-9) m2 V-1 s-1, respectively. The ITP analysis is performed with the driving and detection currents of 50 microA (for 700 s) and 20 microA, respectively. The calibration graphs are rectilinear in the range 25-250 mg l-1 of SO and 50 to 500 mg l-1 of XY. The method is applied to the simultaneous assay of SO and XY in three mass-produced multi-component infusion solutions. Favourable values of the method validation parameters obtained confirm the suitability of the proposed ITP method for the quality control of pharmaceuticals.

Determination of tramadol in various dosage forms by capillary isotachophoresis.

Cationic capillary isotachophoresis (ITP) with conductometric detection has been used for separating and determining milligram amounts of tramadol [2-dimethylaminomethyl-1-(3-methoxyphenyl)-cyclohexanol hydrochloride] (I) in seven commercial mass-produced pharmaceutical preparations. The optimised ITP electrolyte system consisted of 5 mM potassium picolinate + 5 mM picolinic acid (pH 5.25) as the leading electrolyte and 10 mM formic acid as the terminating electrolyte. The driving and detection currents were 50 microA (for 320 s) and 10 microA, respectively (a single analysis took 12-15 min). Under such conditions the effective mobility of I was determined as 24.26 x 10(-9) m2 V(-1) s(-1) (with tetraethylammonium ion as standard); thermodynamic pKa value of I was 9.44 +/- 0.03 (n = 8) as determined by UV spectrophotometry at 25 degrees C and I = 0.01 (NaCl). The calibration graph relating the ITP zone length to the concentration of I was rectilinear (r = 0.99997) in the range 15-180 mg l(-1) of I. The relative standard deviation (RSD) was 0.21% (n = 6) when determining 60 mg l(-1) of I in pure test solution. Sample pre-treatment of the dosage forms involved dilution or extraction of I with water (for suppositories the extraction was carried out in an ultrasonic bath at 40 degrees C for 10 min). The method was suitable for determining 50 or 100 mg ml(-1) of I in injections and drops, 50 mg of I in capsules, and 100 mg of I in suppositories with RSD values 0.4 to 1% (n = 6). According to the validation procedure based on the standard addition technique the recoveries were 97.2-100.1% of I.

Determination of some non-steroid anti-inflammatory drugs by capillary isotachophoresis.

The isotachophoretic (ITP) behaviour and separation of the anti-inflammatory drugs kebuzone (KB), tribuzone (TB) and phenylbutazone (PB) was studied in the operational system of HCl/His (leading electrolyte, LE) and 4-nitrophenol (terminating electrolyte, TE). The effective mobilities were 19.4 x 10(-9) m2 V-1 s-1 for KB, 18.1 x 10(-9) m2 V-1 s-1 for TB and 18.9 x 10(-9) m2 V-1 s-1 for PB when using an optimised system with 10 mM HCl + 40 mM His (pH 6.63) as LE and 10 mM 4-nitrophenol as TE. The calibration graphs were rectilinear (r = 0.9982-0.9996) in the range 20 to 600 mumol 1-1 of KB, TB or PB. The ITP method was used for determining the content of KB, TB or PB in mass-produced pharmaceuticals as tablets, coated tablets, injections, and ointments. The results of the ITP determination were in good agreement with those of standard pharmacopoeial methods.

[Angiotensin converting enzyme inhibitors in controlled hypotension during spinal surgery]. / Inhibitory angiotenzin konvertujícího enzymu v rízené hypotenzi behem spondylochirurgických výkonu.

BACKGROUND: Controlled hypotension is an advantage during spondylosurgical operations: the objective is to achieve a mean arteriae pressure of 8 kPa (60 mm Hg). The most frequently used 0.01% solution of sodium nitroprusside must be increased in some patients to amounts which involve the risk of intoxication. This applies to patients with an increased sympathoadrenal activity and ready mobilization of the renin-angiotensin system. The objective of the present investigation was to test the inhibitor of the angiotensin converting enzyme in hypotension controlled by nitroprusside. METHODS AND RESULTS: To twenty patients before a spondylosurgical operation as premedication angiotensin converting enzyme inhibitor (ACE)--captopril--was administered, 25 mg by the oral route. The control group was formed by 20 patients with spondylosurgery under controlled hypotension with nitroprusside administration. The effect of captopril was manifested by a reduced amount of nitroprusside needed to maintain the median pressure of 8 kPa; in the captopril group 1.073 +/- 0.52 microgram.kg-1.min-1 was used, as compared with 1.786 +/- 1.04 micrograms.min-1 in the control group (p < 0.01). Concurrently monitored values of plasma renin activity were higher in the patients given captopril: 7.352 +/- 5.75 nmol.l-1, as compared with 5.583 +/- 3.73 nmol.l-1 (p < 0.05). CONCLUSIONS: Premedication with ACE inhibitor (captopril), even when administered in small doses via p. o., reduced the sodium nitroprusside consumption by as much as 60%. The elevated plasma renin values were objective evidence of the effect of captopril.

[Initial experience with peroperative autotransfusion in spinal surgery]. / První zkusenosti s peroperacní autotransfúzí ve spondylochirurgii.

The authors describe combinations of anaesthesiological methods which enabled them during extensive spondylosurgical operations in 66 patients to reduce the consumption of homologous blood during operation to 90 ml, on the first day after operation to 300 ml and on the second day after operation to 120 ml. In six patients they used preoperative collection of the patient's own blood, in 45 patients acute normovolaemic haemodilution, in all patients controlled hypotension with sodium nitroprusside to a mean arterial pressure of 8-9 kPa and peroperative collection of blood by means of an autotransfusion apparatus Dideco Stat with a standard programme and yield higher than 50%. During and after peroperative collection they did not record any complications. Lower haemoglobin and haematocrit values and a reduced number of erythrocytes, lower than the lower normal range, during and after operation did not threaten the postoperative course in these patients. The authors draw, however, attention to the rise of the number of leucocytes immediately after operation to 19.7 x 10(9). 1(-1) which is due to their shift into the final product. Solution of this phenomenon which can produce ARDS is according to the authors the use of a programme different from the standard one.