Quantitative Investigation of Terbinafine Hydrochloride Absorption into a Living Skin Equivalent Model by MALDI-MSI.

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods.

Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity.

The l-alanine dehydrogenase of Bacillus subtilis (BasAlaDH), which is strictly dependent on NADH as redox cofactor, efficiently catalyzes the reductive amination of pyruvate to l-alanine using ammonia as amino group donor. To enable application of BasAlaDH as regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases, we alterated its cofactor specificity from NADH to NADPH via protein engineering. By introducing two amino acid exchanges, D196A and L197R, high catalytic efficiency for NADPH was achieved, with kcat /KM  = 54.1 µM-1  Min-1 (KM  = 32 ± 3 µM; kcat  = 1,730 ± 39 Min-1 ), almost the same as the wild-type enzyme for NADH (kcat /KM  = 59.9 µM-1  Min-1 ; KM  = 14 ± 2 µM; kcat  = 838 ± 21 Min-1 ). Conversely, recognition of NADH was much diminished in the mutated enzyme (kcat /KM  = 3 µM-1  Min-1 ). BasAlaDH(D196A/L197R) was applied in a coupled oxidation/transamination reaction of the chiral dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans ω-aminotransferase, thus allowing recycling of the two cosubstrates NADP+ and l-Ala. An excellent cofactor regeneration with recycling factors of 33 for NADP+ and 13 for l-Ala was observed with the engineered BasAlaDH in a small-scale biocatalysis experiment. This opens a biocatalytic route to novel building blocks for industrial high-performance polymers.

In vitro-in vivo correlation in skin permeation.

PURPOSE: In vitro skin permeation studies have been used extensively in the development and optimisation of delivery of actives in vivo. However, there are few reported correlations of such in vitro studies with in vivo data. The aim of this study was to investigate the skin permeation of a model active, niacinamide, both in vitro and in vivo. METHODS: Conventional diffusion cell studies were conducted in human skin to determine niacinamide permeation from a range of vehicles which included dimethyl isosorbide (DMI), propylene glycol (PG), propylene glycol monolaurate (PGML), N-methyl 2-pyrrolidone (NMP), Miglyol 812N® (MG), and mineral oil (MO). Single, binary or ternary systems were examined. The same vehicles were subsequently examined to investigate niacinamide delivery in vivo. For this proof-of-concept study one donor was used for the in vitro studies and one volunteer for the in vivo investigations to minimise biovariability. Analysis of in vitro samples was conducted using HPLC and in vivo uptake of niacinamide was evaluated using Confocal Raman spectroscopy (CRS). RESULTS: The amount of niacinamide permeated through skin in vitro was linearly proportional to the intensity of the niacinamide signal determined in the stratum corneum in vivo. A good correlation was observed between the signal intensities of selected vehicles and niacinamide signal intensity. CONCLUSIONS: The findings provide further support for the use of CRS to monitor drug delivery into and across the skin. In addition, the results highlight the critical role of the vehicle and its disposition in skin for effective dermal delivery.

Isosorbide-based cholinesterase inhibitors; replacement of 5-ester groups leading to increased stability.

Isosorbide-2-carbamate-5-esters are highly potent and selective butyrylcholinesterase inhibitors with potential utility in the treatment of Alzheimer's Disease (AD). They are stable in human plasma but in mouse plasma they undergo hydrolysis at the 5-ester group potentially attenuating in vivo potency. In this paper we explore the role of the 5-position in modulating potency. The focus of the study was to increase metabolic stability while preserving potency and selectivity. Dicarbamates and 5-keto derivatives were markedly less potent than the ester class. The 2-benzylcarbamate-5-benzyl ether was found to be potent (IC(50) 52 nM) and stable in the presence of mouse plasma and liver homogenate. The compound produces sustained moderate inhibition of mouse butyrylcholinesterase at 1mg/kg, IP.

Isosorbide-2-benzyl carbamate-5-salicylate, a peripheral anionic site binding subnanomolar selective butyrylcholinesterase inhibitor.

Isosorbide-2-benzyl carbamate-5-benzoate is a highly potent and selective BuChE inhibitor. Meanwhile, isosorbide-2-aspirinate-5-salicylate is a highly effective aspirin prodrug that relies on the salicylate portion to interact productively with human BuChE. By integrating the salicylate group into the carbamate design, we have produced isosorbide-2-benzyl carbamate-5-salicylate, an inhibitor of high potency (150 pM) and selectivity for human BuChE over AChE (666000) and CES2 (23000). Modeling and mutant studies indicate that it achieves its exceptional potency because of an interaction with the polar D70/Y332 cluster in the PAS of BuChE in addition to pseudosubstrate interactions with the active site.

Novel isosorbide-based substrates for human butyrylcholinesterase.

Butyrylcholinesterase [EC 3.1.1.8] present widely in mammalian tissue does not have a precisely defined biological function or known endogenous substrate. However, it plays an important role in the detoxification of certain xenobiotics and is an established vector for the systemic liberation of other drugs from their prodrugs. While investigating a series of isosorbide-based prodrugs, we discovered that BuChE catalyses the hydrolysis of esters of the simple sugar isosorbide with unusually rapidity and in some cases with remarkable regioselectivity. In this study, a series of isosorbide esters were synthesised and their rates of hydrolysis measured by HPLC following incubation in diluted plasma solution. In general, little hydrolysis of the 5-ester group could be observed but the 2-ester group was usually hydrolysed very rapidly and the hydrolysis rate exhibited an unusual dependence on the identity of the 5-group. The results indicate that while the 5-ester group is not itself hydrolysed it is important for productive binding in isosorbide diesters.

Isosorbide-based aspirin prodrugs. II. Hydrolysis kinetics of isosorbide diaspirinate.

Aspirin prodrugs have been intensively investigated in an effort to produce compounds with lower gastric toxicity, greater stability or enhanced percutaneous absorption, relative to aspirin. This report describes the hydrolysis kinetics and aspirin release characteristics of isosorbide diaspirinate (ISDA), the aspirin diester of isosorbide. ISDA underwent rapid hydrolysis when incubated in phosphate buffered human plasma solutions (pH 7.4) at 37 degrees C, producing appreciable quantities of aspirin. In 30% human plasma solution the half-life was 1.1 min and 61% aspirin was liberated relative to the initial ester concentration. The hydrolysis kinetics of ISDA were monitored in aqueous solution at 37 degrees C over the pH range 1.03-9.4. The aqueous hydrolysis followed pseudo-first-order kinetics over several half-lives at all pH values, resulting in a U-shaped pH rate profile. Salicylate esters and salicylic acid were formed during these processes. The hydrolysis characteristics of ISDA were also investigated in pH 7.4 phosphate buffered solutions containing alpha-chymotrypsin [EC 3.1.1.1] (t(1/2)=200.9 min), carboxyl esterase [EC 3.1.1.1] (t(1/2)=31.5 min), human serum albumin (t(1/2)=603 min), purified human serum butyrylcholinesterase [EC 3.1.1.8] (80 micro g/ml; t(1/2)=9.4 min; 55% aspirin), purified horse serum butyrylcholinesterase (100 micro g/ml; t(1/2)=1.85 min;11% aspirin) and in 10% human plasma solution in the presence of physostigmine (3 micro M). The results indicate that a specific enzyme present in human plasma, probably human butyrylcholinesterase, catalyses aspirin release from isosorbide diaspirinate.

A highly efficient asymmetric synthesis of optically active alpha, gamma-substituted gamma-butyrolactones using a chiral auxiliary derived from isosorbide.

Using an easily accessible and inexpensive chiral auxiliary derived from isosorbide, optically active alpha,gamma-substituted gamma-butyrolactones were obtained in high enantiomeric purity (up to >99% ee for trans) by the SmI(2)-induced reductive coupling of chiral methacrylate 7 with ketones in the presence of (-)-sultam as a proton source.

Metabolic fact of 14C-isosorbide 5-mononitrate in humans.

Single oral doses of 20 mg of the carbon-14 labelled form of the antianginal drug isosorbide 5-mononitrate (5-ISMN, Elantan) were essentially completely absorbed and excreted fairly rapidly in the urine. Means of 24, 52, 78, 93 and 96% dose were excreted during 6, 12, 24, 48 and 120 h, respectively. Concentrations of 14C reached peak levels at about 1-2 h when about 86% of the 14C was associated with the parent drug, 5-ISMN (peak mean level 430 ng/ml), and the remainder mainly with the pharmacologically-inactive denitrated product isosorbide. Because the plasma (and urinary) half-life of isosorbide was longer (about 8-9 h) than that of 5-ISMN (about 4.5 h), the proportions of the former in plasma increased relative to the latter. Concentrations of 14C in whole-blood and plasma were similar, implying that 5-ISMN diffused into blood cells. Concentrations of 5-ISMN in saliva and plasma were almost identical, presumably because of the almost negligible plasma protein binding of the drug (less than 5%). At least five metabolites of 5-ISMN were detected in urine - these were isosorbide (about 37% dose), conjugated material (about 25% dose) presumably mainly 5-ISMN-glucuronide, sorbitol (about 7% dose), the parent drug 5-ISMN (about 2% dose) and two unidentified metabolites (about 7 and 4% dose, respectively). The conjugated material was excreted in the urine relatively more rapidly than the denitrated product, isosorbide.

Determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by high-performance liquid chromatography-thermal energy analysis.

An accurate and sensitive method for the simultaneous determination of isosorbide dinitrate and its 2- and 5-mononitrates in human plasma has been developed. Following extraction of 3.0 ml of plasma with 12.0 ml of dichloromethane-ethyl acetate (1:1) the extract is subjected to high-performance liquid chromatography employing a Zorbax NH2 column. The eluent stream is introduced into a thermal energy analyser, employing chemiluminescence as a specific means of detection. The minimum quantifiable level of the compound in plasma is 200 pg/ml allowing the quantitation of isosorbide dinitrate in human plasma following single oral administration. Nitroglycerin is employed as internal standard.