Antonio Lopalco

Mechanism of Decarboxylation of Pyruvic Acid in the Presence of Hydrogen Peroxide.

The purpose of this work was to probe the rate and mechanism of rapid decarboxylation of pyruvic acid in the presence of hydrogen peroxide (H2O2) to acetic acid and carbon dioxide over the pH range 2-9 at 25 °C, utilizing UV spectrophotometry, high performance liquid chromatography (HPLC), and proton and carbon nuclear magnetic resonance spectrometry ((1)H, (13)C-NMR). Changes in UV absorbance at 220 nm were used to determine the kinetics as the reaction was too fast to follow by HPLC or NMR in much of the pH range. The rate constants for the reaction were determined in the presence of molar excess of H2O2 resulting in pseudo first-order kinetics. No buffer catalysis was observed. The calculated second-order rate constants for the reaction followed a sigmoidal shape with pH-independent regions below pH 3 and above pH 7 but increased between pH 4 and 6. Between pH 4 and 9, the results were in agreement with a change from rate-determining nucleophilic attack of the deprotonated peroxide species, HOO(-), on the α-carbonyl group followed by rapid decarboxylation at pH values below 6 to rate-determining decarboxylation above pH 7. The addition of H2O2 to ethyl pyruvate was also characterized.

Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood-brain barrier and human placental trophoblast.

Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer® RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood–brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140–170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy.

Spray-dried mucoadhesives for intravesical drug delivery using N-acetylcysteine- and glutathione-glycol chitosan conjugates.

UNLABELLED This work describes N-acetylcysteine (NAC)- and glutathione (GSH)-glycol chitosan (GC) polymer conjugates engineered as potential platform useful to formulate micro-(MP) and nano-(NP) particles via spray-drying techniques. These conjugates are mucoadhesive over the range of urine pH, 5.0-7.0, which makes them advantageous for intravesical drug delivery and treatment of local bladder diseases. NAC- and GSH-GC conjugates were generated with a synthetic approach optimizing reaction times and purification in order to minimize the oxidation of thiol groups. In this way, the resulting amount of free thiol groups immobilized per gram of NAC- and GSH-GC conjugates was 6.3 and 3.6mmol, respectively. These polymers were completely characterized by molecular weight, surface sulfur content, solubility at different pH values, substitution and swelling degree. Mucoadhesion properties were evaluated in artificial urine by turbidimetric and zeta (ζ)-potential measurements demonstrating good mucoadhesion properties, in particular for NAC-GC at pH 5.0. Starting from the thiolated polymers, MP and NP were prepared using both the Büchi B-191 and Nano Büchi B-90 spray dryers, respectively. The resulting two formulations were evaluated for yield, size, oxidation of thiol groups and ex-vivo mucoadhesion. The new spray drying technique provided NP of suitable size (<1μm) for catheter administration, low degree of oxidation, and sufficient mucoadhesion property with 9% and 18% of GSH- and NAC-GC based NP retained on pig mucosa bladder after 3h of exposure, respectively. STATEMENT OF SIGNIFICANCE The aim of the present study was first to optimize the synthesis of NAC-GC and GSH-GC, and preserve the oxidation state of the thiol moieties by introducing several optimizations of the already reported synthetic procedures that increase the mucoadhesive properties and avoid pH-dependent aggregation. Second, starting from these optimized thiomers, we studied the feasibility of manufacturing MP and NP by spray-drying techniques. The aim of this second step was to produce mucoadhesive drug delivery systems of adequate size for vesical administration by catheter, and comparable mucoadhesive properties with respect to the processed polymers, avoiding thiolic oxidation during the formulation. MP with acceptable size produced by spray-dryer Büchi B-191 were compared with NP made with the apparatus Nano Büchi B-90.

Determination of pKa and Hydration Constants for a Series of α-Keto-Carboxylic Acids Using Nuclear Magnetic Resonance Spectrometry

The determination of the acid-base dissociation constants, and thus the pKa values, of α-keto acids such as pyruvic acid is complex because of the existence of these acids in their hydrated and nonhydrated or oxo state. Equilibria involved in the hydration and dehydration of the α-keto group of pyruvic acid and three other α-keto acids, 3-methyl-2-oxobutanoic acid, 4-methyl-2-oxopentanoic acid, and 2-oxo-2-phenylacetic acid, were investigated by proton and carbon nuclear magnetic resonance spectrometry, at constant ionic strength, 0.15, and 25 °C. Dissociation constants for the oxo (pKa(oxo)) and hydrated (pKa(hyd)) acids of each compound were estimated from the change in the degree of hydration with changes in pH and directly from the changes in chemical shifts of various hydrogen and carbons nuclei with pH. α-Keto acids showed greater hydration in their acidic forms than their carboxylate forms. The degree of hydration was sensitive to steric and electronic/resonance factors. As expected, the oxo forms of the acids were stronger acids compared with their hydrated analogs, and their dissociation constants were also sensitive to steric and electronic factors.

Effect of Molecular Structure on the Relative Hydrogen Peroxide Scavenging Ability of Some α-Keto Carboxylic Acids

The α-keto carboxylic acid, pyruvic acid (1) was found to be a very effective peroxide scavenger but is subject to an aldol-like self-condensation/polymerization reaction. The purpose of this study was to evaluate the hydrogen peroxide, H2O2, scavenging ability of 3-methyl-2-oxobutanoic acid (2), 4-methyl-2-oxopentanoic acid (3), and 2-oxo-2-phenylacetic acid (phenylglyoxylic acid, 4) in the pH range 2-9 at 25°C and the effect of molecular structure on the relative reactivity. The reaction with H2O2 was followed by UV spectrophotometry at 220 or 260 nm and high-performance liquid chromatography. Pseudo-first order, buffer-independent decarboxylation kinetics were observed in the presence of molar excess H2O2. The second-order rate constants for 2-4 followed a sigmoidal shape and mechanism similar to pyruvic acid. Pyruvic acid was a superior H2O2 scavenger to 2-4 over the pH range 2-9 but 4 was more reactive than 2 and 3 at pH values above 6. There was a qualitative correlation between the degree of keto-group hydration and reactivity of the acids in the pH range 4-6 while the data above pH 7 suggested that the intrinsic decarboxylation step for 4 was faster than for pyruvic acid. Differences in reactivity to molecular structure were analyzed.

New ethanol and propylene glycol free gel formulations containing a minoxidil-methyl-β-cyclodextrin complex as promising tools for alopecia treatment

Abstract New topical totally aqueous formulations that improve the low water solubility of minoxidil and realize an adequate permeability of drug in the skin are proposed. These formulations are lacking in propylene glycol and alcohol that are the principal irritant ingredients present in minoxidil commercial solutions. In order to enhance poor water solubility of minoxidil randomly methyl-β-cyclodextrin was used, and four hydrogels such as, calcium alginate, sodium alginate, carbopol 934 and hydroxyethylcellulose were utilized to ensure a prolonged time of contact with the scalp. The inclusion complex minoxidil/methyl-β-cyclodextrin with a molar ratio 1:1 was obtained by freeze drying and evaluated by NMR, FT-IR and DSC analysis. An apparent stability constant of formed inclusion complex was calculated by phase solubility diagram and its value was 400 M−1. The solid inclusion complex was used to prepare gel formulations with similar dose to minoxidil commercial solution. The gels were evaluated for various technological parameters including rheological behavior, in vitro drug release and ex vivo permeation through pig skin. The best performance was observed for the calcium alginate formulation.

TSPO Ligand-Methotrexate Prodrug Conjugates: Design, Synthesis, and Biological Evaluation

The 18-kDa translocator protein (TSPO) is a potential mitochondrial target for drug delivery to tumors overexpressing TSPO, including brain cancers, and selective TSPO ligands have been successfully used to selectively deliver drugs into the target. Methotrexate (MTX) is an anticancer drug of choice for the treatment of several cancers, but its permeability through the blood brain barrier (BBB) is poor, making it unsuitable for the treatment of brain tumors. Therefore, in this study, MTX was selected to achieve two TSPO ligand-MTX conjugates (TSPO ligand α-MTX and TSPO ligand γ-MTX), potentially useful for the treatment of TSPO-rich cancers, including brain tumors. In this work, we have presented the synthesis, the physicochemical characterizations, as well as the in vitro stabilities of the new TSPO ligand-MTX conjugates. The binding affinity for TSPO and the selectivity versus central-type benzodiazepine receptor (CBR) was also investigated. The cytotoxicity of prepared conjugates was evaluated on MTX-sensitive human and rat glioma cell lines overexpressing TSPO. The estimated coefficients of lipophilicity and the stability studies of the conjugates confirm that the synthesized molecules are stable enough in buffer solution at pH 7.4, as well in physiological medium, and show an increased lipophilicity compared to the MTX, compatible with a likely ability to cross the blood brain barrier. The latter feature of two TSPO ligand-MTX conjugates was also confirmed by in vitro permeability studies conducted on Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) monolayers. TSPO ligand-MTX conjugates have shown to possess a high binding affinity for TSPO, with IC50 values ranging from 7.2 to 40.3 nM, and exhibited marked toxicity against glioma cells overexpressing TSPO, in comparison with the parent drug MTX.

Isolation, Solubility, and Characterization of D-Mannitol Esters of 4-Methoxybenzeneboronic Acid

The purpose of this study was to determine the aqueous solubility of a model phenyl boronic acid, 4-methoxybenzeneboronic acid, as a function of pH both in the absence and in the presence of varying D-mannitol concentration. Solid isolated D-mannitol esters were characterized by differential scanning calorimetry, thermogravimetric analysis, powder X-ray diffraction, and single-crystal X-ray studies, and the boronic acid-to-D-mannitol ratio was quantified by HPLC. Hydrolysis of the monoester was studied using UV spectral differences between the monoester and the parent boronic acid. Two D-mannitol esters of 4-methoxybenzeneboronic acid were isolated. The triboronate ester was very insoluble whereas a symmetrical monoboronate monohydrate was also less soluble than the parent. Both esters were crystalline. The monoboronate monohydrate was, however, more soluble than the parent at alkaline pH values due to its lower pKa value (6.53) compared to the parent acid (9.41). Hydrolysis of the monoboronate was extremely fast when even small amount of water was added to dry acetonitrile solutions of the ester. The hydrolysis was buffer concentration dependent and apparent pH sensitive with hydrolysis accelerated by acid. Implications affecting the formulation of future boronic acid drugs are discussed.

Mechanism of Degradation of an α-Keto-Epoxide, a Model for the Warhead for Various Proteasome Inhibitor Anticancer Agents

The anticancer agent, carfilzomib, has a unique α-keto-epoxide warhead. The model α-keto-epoxide, N-((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)pivalamide (1), along with a few of its degradation products was synthesized and studied. The kinetics of hydrolysis and identification of some of the degradation products of 1 were performed at pH values 2, 4, 5, 7, and 8 at 25°C, 40°C, and 60°C and followed by HPLC and liquid chromatography-mass spectroscopy, respectively. 1 degraded independent of pH between pH values 4-7 but showed some acid catalysis at pH 2 and base catalysis at pH 8. Energy of activation, Ea, values progressed from 16.8 ± 0.1 at pH 2 to 20.3 ± 0.1 kcal/mole at pH 8. The major initial degradation products in the pH range 4-5 were the S,R diol (hydrolysis of the epoxide), and S,R chlorohydrin (in the presence of chloride ions). At pH 7-8, the major products were the R,R diastereomer and the S,R and R,R diols. At pH 2, additional unidentified products were seen with relative retention times of 0.28, 0.30, 0.33, and 0.35 and masses equivalent to the diols. The study of 1 provides insight into the degradation of future drugs that use an α-keto-epoxide functional group.

Origins, and formulation implications, of the pKa difference between boronic acids and their esters: A density functional theory study

ABSTRACT The purpose of this study is to try to identify the etiology and formulation implications of the significant pKa drop in aqueous solution that occurs when boronic acids are reversibly esterified by reaction with alcohols, especially 1,2‐diols. Experimental studies have shown that conversion of a boronic acid to a boronic acid ester, both Lewis acids, is accompanied by an increase in the acidity, that is, a lowering of the pKa value in aqueous solution. The drop in pKa value has significant implications for the formulation of boronic acid‐based drugs. Specifically, in the presence of 1,2‐diols, a &Dgr;pKa, a drop, of about 3 pKa units has been observed for several boronic acids, including boron‐based drugs that have demonstrated value as active pharmaceutical ingredients. Boric acid itself shows an even larger drop of about 4units. Here, electronic structure calculations are used to investigate the origins of this pKa change. Specifically, density functional theory (DFT) is used to predict the &Dgr;pKa for a model boronic acid, methylboronic acid, to two of its esters, the dimethyl ester and the cyclic ester with ethylene glycol. The approach is validated by accurately predicting the experimentally observed increased acidity of the cyclic boronic acid ester relative to the corresponding boronic acid. The origins of the acidity difference are examined in detail. Analysis shows that the primary origin is an electronic effect with a smaller contribution due to the reduced structural flexibility of the boronic ester.