Massimo Pregnolato

Disposition of high-dose busulfan in pediatric patients undergoing bone marrow transplantation

We studied the pharmacokinetics of busulfan (16 mg/kg) in 16 pediatric patients affected by malignant and nonmalignant disorders between 6 months and 19 years of age (mean ± SD, 5.7 ± 6.5 years) who were undergoing allogeneic (15 patients) and autologous (one patient) bone marrow transplantation. In all children, the conditioning regimen consisted of busulfan given orally at a dose of 1 mg/kg every 6 hours for 16 doses (total dose, 16 mg/kg), associated with other drugs. The pharmacokinetics of busulfan was studied during the 6‐hour dosing interval on the third day of therapy by use of a high‐performance liquid chromatographie assay. The value for the time to reach maximum concentration, expressed as mean ± SD, was 1.1 ± 0.5 hour; maximum concentration was 609.6 ± 225.3 ng/ml; steady‐state concentration was 358.9 ± 135.5 ng/ml; area under the plasma concentration–time curve was 2153.6 ± 813.1 ng · hr/ml; oral clearance was 0.535 ± 0.226 L/hr/kg; and half‐life was 2.4 ± 0.8 hours. Age‐related differences in busulfan disposition were observed. The mean busulfan oral clearance in a group of 10 patients with an age range from 6 months to 3 years was 0.619 L/hr/kg, whereas six patients whose ages ranged from 7 to 19 years had a oral clearance of 0.396 L/hr/kg. The half‐lives for busulfan during infancy decrease continuously until early childhood but were prolonged in older children. No significant relationship between systemic exposure to busulfan and drug effect was observed.

Selective Interaction of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Nonnucleoside Inhibitor Efavirenz and Its Thio-Substituted Analog with Different Enzyme-Substrate Complexes

ABSTRACT Accumulating data have brought the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) into the forefront of antiretroviral therapy. Among the emerging compounds in this class, a particularly attractive one is efavirenz (Sustiva), recently approved for clinical use by the U.S. Food and Drug Administration. In the present study, the equilibrium dissociation constants for efavirenz binding to the different catalytic forms of human immunodeficiency virus type 1 RT as well as the association and dissociation rates have been determined using a steady-state kinetic approach. In addition, the same enzymological analysis has been extended to the thio-substituted analog, sefavirenz, which showed comparable activity in vitro against RT. Both compounds have been found to act as purely uncompetitive inhibitors at low drug concentrations (5 to 50 nM) and as mixed noncompetitive inhibitors at higher doses (50 to 500 nM). This behavior can be interpreted in terms of the relative affinities for the different catalytic forms of the enzyme. Both efavirenz and sefavirenz showed increasing affinities for the different forms of RT in the following order: free enzyme < (i.e., bound with lower affinity) binary RT–template-primer (TP) complex < ternary RT-TP-deoxynucleoside triphosphate (dNTP) complex. The rate of binding of the two inhibitors to the different enzyme-substrate complexes was well below the diffusion limit (on the order of 104M−1 s−1); however, both inhibitors, when bound to the ternary RT-TP-dNTP complex, showed very low dissociation rates, on the order of 10−4 s−1 for both compounds, typical of tightly binding inhibitors. Thus, efavirenz and its thio-substituted derivative sefavirenz appear to be peculiar in their mechanism of action, being selective tightly binding inhibitors of the ternary RT-TP-dNTP complex. Efavirenz is the first clinically approved NNRTI to show this property.

High-performance Liquid Chromatography Method for Analyzing the Antiretroviral Agent Efavirenz in Human Plasma

Efavirenz (EFV, DMP-266) is a new antiretroviral agent belonging to the class of nonnucleoside reverse transcriptase inhibitors. It has recently been approved by the Food and Drug Administration in management of human immunodeficiency virus (HIV). Preliminary pharmacokinetic studies on EFV in healthy volunteers show that the drug may influence the metabolism of protease inhibitors. For the determination of EFV in human plasma, a validated and specific reverse-phase high-performance liquid chromatography (HPLC) method, with UV detection, was developed. We used 100 microL plasma sample for a liquid-liquid extraction with diethyl ether after basification. The mobile phase was a mixture of acetonitrile and water, pumped at a flow rate of 1.2 mL/min. Ultraviolet detection was carried out at a wavelength of 247 nm. Retention times for EFV and internal standard (IS) were 5.3 and 4.5 minutes, respectively, and there was no chromatographic interference from other commonly administered drugs. The limit of detection was 100 ng/mL. The described assay is a rapid and accurate method for measurement of EFV in plasma: the easy preparation and small sample size makes this assay highly suitable for pharmacokinetic studies and routine clinical analysis in patients with HIV. In addition, the reproducibility of the method is only moderately increased by including IS, so analyzing without IS may be an alternative.

3H-[1,2]Dithiolo[3,4-b]pyridine-3-thione and its derivatives Synthesis and antimicrobial activity

A series of 2-substituted isothiazolo[5,4-b]pyridine-3(2H)-thiones, isothiazolo[5,4-b]pyridin-3(2H)-ones, N-substituted 2-sulfanylnicotinamides and the corresponding carbothioamide derivatives were synthesized and evaluated for their antimicrobial activity against several strains of Gram+ and Gram- bacteria and fungi. Chemical syntheses were resumed into a comprehensive cyclic route that enables the reversible conversion for each derivative of the series considered. Among the tested compounds the N-(aralkyl)-2-sulfanylnicotinamides show the highest fungitoxicity (MIC = 1.25-5 microg/ml). The best activity towards Gram-positive bacteria was in the range of 2.5-5 microg/ml. Activity against Gram-negative bacteria was generally very poor for all compounds.

Enantioselective enzymatic hydrolysis of racemic glycidyl esters by using immobilized porcine pancreas lipase with improved catalytic properties

The crude porcine pancreas lipase (PPL) extract is a mixture of several proteins (mainly lipases and esterases). In order to develop enzymatic catalysts with good catalytic properties for hydrolytic enantioselective reactions in aqueous homogeneous medium, we studied the immobilization of the different enzymes contained in the crude PPL extracts by selective sequential adsorption on hydrophobic supports bearing octyl, octadecyl and phenyl groups. Some minor proteins were selectively adsorbed on octyl and octadecyl supports while the most abundant lipase was adsorbed on the support bearing phenyl groups. The enantioselectivity of the different lipase derivatives were tested considering the hydrolysis of esters of 1,2-epoxi-1-propanol (glycidol). The most abundant lipase contained in the commercial crude PPL extract resulted almost inactive while some lipases contained in low concentrations displayed high activities and enantioselectivities. The most interesting results were obtained with a 28-kDa protein selectively adsorbed on octyl-agarose. With this enzyme derivative, the residual butyric ester of glycidol was recovered with 96% enantiomeric excess at 55% conversion.

Quantum neurophysics: From non-living matter to quantum neurobiology and psychopathology

The concepts of quantum brain, quantum mind and quantum consciousness have been increasingly gaining currency in recent years, both in scientific papers and in the popular press. In fact, the concept of the quantum brain is a general framework. Included in it are basically four main sub-headings. These are often incorrectly used interchangeably. The first of these and the one which started the quantum mind/consciousness debate was the place of consciousness in the problem of measurement in quantum mechanics. Debate on the problem of quantum measurement and about the place of the conscious observer has lasted almost a century. One solution to this problem is that the participation of a conscious observer in the experiment will radically change our understanding of the universe and our relationship with the outside world. The second topic is that of quantum biology. This topic has become a popular field of research, especially in the last decade. It concerns whether or not the rules of quantum physics operate in biological structures. It has been shown in the latest research on photosynthesis, the sense of smell and magnetic direction finding in animals that the laws of quantum physics may operate in warm-wet-noisy biological structures. The third sub-heading is quantum neurobiology. This topic has not yet gained wide acceptance and is still in its early stages. Its primary purpose is directed to understand whether the laws of quantum physics are effective in the biology of the nervous system or not. A further step in brain neurobiology, toward the understanding of consciousness formation, is the research of quantum laws effects upon neural network functions. The fourth and final topic is quantum psychopathology. This topic takes its basis and its support from quantum neurobiology. It comes from the idea that if quantum physics is involved in the normal working of the brain, diseased conditions of the brain such as depression, anxiety, dementia, schizophrenia and hallucinations can be explained by quantum physical pathology. In this article, these topics will be reviewed in a general framework, and for the first time a general classification will be made for the quantum brain theory.

Chemoenzymatic one-pot synthesis of cefazolin from cephalosporin C in fully aqueous medium, involving three consecutive biotransformations catalyzed by D-aminoacid oxidase, glutaryl acylase and penicillin G acylase

Abstract A new chemoenzymatic synthesis of Cefazolin through the correct assembly of three biotransformations catalysed by D-aminoacid oxidase, glutaryl acylase and penicillin G acylase is described. This multienzymatic synthesis has been performed from the natural Cephalosporin C in fully aqueous medium without intermediate purification stages. Almost quantitative yields have been achieved in all the enzymatic reactions.

Deuterated internal standards for gas chromatographic-mass spectrometric analysis of polar organophosphorus pesticides in water samples

It is essential to know whether drinking water contains any pesticides up to concentrations close to the European Union limit of 0.1 microgram/l, in order to protect the population. Mass spectrometry (MS) using a suitable internal standard (I.S.) should improve the quantitative analysis of pesticides, the I.S. circumventing loss of compounds during the analytical procedure and correcting the analytical variability. In this study we verified this assumption in GC-MS, synthesising specific internal standards for four organophosphorus pesticides with poor stability, comparing the performances with and without the I.S.

Enzymatic synthesis of cephalosporins. The immobilized acylase from Arthrobacter viscosus: a new useful biocatalyst.

The acylase from Arthrobacter viscosus was immobilized, studied in the enzymatic synthesis of some cephalosporins by kinetically controlled N-acylation (kcNa) of different cephem nuclei, and compared with the penicillin G acylase (PGA) from Escherichia coli. The reaction outcomes were dependent on the acylase microbial source and on the type of immobilization support. Generally, both enzymes, when immobilized onto hydrophilic resins such as glyoxyl-agarose (activated with aldehyde groups), displayed higher synthetic performances in comparison with hydrophobic acrylic epoxy-supports like Eupergit C. The kcNa of 7-amino cephalosporanic acid catalyzed by A. viscosus immobilized on glyoxyl-agarose afforded a quantitative conversion in 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ3-cephem-4-carboxylic acid, a useful intermediate for the synthesis of Cefamandole and Cefonicid. Similar results were obtained in the synthesis of these cephalosporins by direct acylation of the corresponding 3′-functionalized nucleus. In these reactions, A. viscosus displayed higher synthetic performances than the PGA from E. coli.

Potentiation of Inhibition of Wild-Type and Mutant Human Immunodeficiency Virus Type 1 Reverse Transcriptases by Combinations of Nonnucleoside Inhibitors and d- and l-(β)-Dideoxynucleoside Triphosphate Analogs

ABSTRACT Combinations of reverse transcriptase (RT) inhibitors are currently used in anti-human immunodeficiency virus therapy in order to prevent or delay the emergence of resistant virus and to improve the efficacy against viral enzymes carrying resistance mutations. Drug-drug interactions can result in either positive (additive or synergistic inhibition) or adverse (antagonistic interaction, synergistic toxicity) effects. Elucidation of the nature of drug interaction would help to rationalize the choice of antiretroviral agents to be used in combination. In this study, different combinations of nucleoside and nonnucleoside inhibitors, including d- andl-(β)-deoxy- and -dideoxynucleoside triphosphate analogues, have been tested in in vitro RT assays against either recombinant wild-type RT or RT bearing clinically relevant nonnucleoside inhibitor resistance mutations (L100I, K103N, Y181I), and the nature of the interaction (either synergistic or antagonistic) of these associations was evaluated. The results showed that (i) synergy of a combination was not always equally influenced by the individual agents utilized, (ii) a synergistic combination could improve the sensitivity profile of a drug-resistant mutant enzyme to the single agents utilized, (iii) l-(β)-enantiomers of nucleoside RT inhibitors were synergistic when combined with nonnucleoside RT inhibitors, and (iv) inter- and intracombination comparisons of the relative potencies of each drug could be used to highlight the different contributions of each drug to the observed synergy.