Abstract  The role of the vehicle in the percutaneous absorption of clindamycin hydrochloride has been studied. Fourteen vehicles have been examined for their ability (1) to adequately solubilize clindamycin and (2) to effect penetration of the drug into the skin. The solubility of clindamycin was found to be good in ten of the fourteen vehicles studied, with a drug concentration of greater than 1.0% being achieved. A vehicular water content of greater than 20% or the presence of another suitable cosolvent appeared to be necessary for adequate solubilization of drug. Percutaneous absorption varied greatly among the vehicles, ranging from 0.7% to 12.9% of the applied dose in 24 hours. The vehicles effecting the greatest clindamycin absorption contained the penetration enhancers dimethylsulfoxide or N-methyl-2-pyrrolidone. Overall, the vehicles studied varied a hundredfold with respect to clindamycin bioavailability. Correlation of these data with other literature data suggests a possible relationship between clindamycin bioavailability and clinical efficacy in the treatment of acne and, therefore, argues against extemporaneous compounding of this drug in the absence of bioavailability data.

Abstract  There is an increasing commercial demand for naturally sourced indigo that meets the purity standards set by the synthetic product. This study concerns the indigo made from leaves of woad (Isatis tinctoria L.), and in particular its interaction with particulate impurities arising from soil and plant materials. Also, a more reliable method using N-methyl-2-pyrrolidone has been developed for the spectrophotometric determination of indigo. In a novel application of fluorescence spectroscopy, indoxyl intermediates in indigo formation are shown to be stable for minutes. The main indigo precursor from woad can be adsorbed onto Amberlite XAD16 in conformity with a Langmuir isotherm, but indigo precursors break down on this and other resin beads to yield indigo and red compounds. Indigo made from indoxyl acetate aggregates into particles, the size distribution of which can be modified by the inclusion of a fine dispersion of calcium hydroxide. Bright field microscopy of indigo products made under defined conditions and scanning electron microscopy combined with energy-dispersive X-ray analysis reveal the relationship of indigo with particulate materials. A model illustrating the interaction of indigo with particulate contaminants is developed on the basis of the results obtained, and recommendations are made for improving the purity of natural indigo.

Abstract  Regulation of RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclast differentiation is of current interest in the development of antiresorptive agents. Osteoclasts are multinucleated cells that play a crucial role in bone resorption. In this study, we investigated the effects of N-methylpyrrolidone (NMP) on the regulation of RANKL-induced osteoclastogenesis. NMP inhibited RANKL-induced tartrate-resistant acid phosphatase activity and the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. The RANKL-induced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos, which are key transcription factors for osteoclastogenesis, was also reduced by treatment with NMP. Furthermore, NMP induced disruption of the actin rings and decreased the mRNAs of cathepsin K and MMP-9 (matrix metalloproteinase-9), both involved in bone resorption. Taken together, these results suggest that NMP inhibits osteoclast differentiation and attenuates bone resorption. Therefore, NMP could prove useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption.

Abstract  BACKGROUND: Weight gain is one of the consequences of estrogen deficiency and constitutes a major health problem. The present study highlights the effects of N-methyl pyrrolidone (NMP) on adipogenesis in osteoporosis induced by estrogen deficiency in an ovariectomized rat model.

RESULTS: Ovariectomy resulted in body weight gain, increased femoral marrow adipocytes, and hypertrophic adipocytes in white adipose tissue, distorted serum leptin, and TNF-α and PPARγ levels. Treatment with NMP normalized these parameters similar to the control group. In vitro, NMP inhibited the differentiation of 3T3-L1 pre-adipocytes and hMSCs, indicating its anti-adipogenic effect. Moreover, PPARγ was significantly reduced with NMP treatment in in vivo and in vitro experiments. NMP inhibited BRD2 and BRD4 binding in an AlphaScreen assay, with an IC50 of 3 and 4 mM, respectively. The effect of NMP was consistent with its role as a bromodomain inhibitor.

CONCLUSIONS: Our data indicates that NMP inhibits the adipogenic effect of estrogen deficiency at the level of PPARγ expression by BRD4 inhibition.

Abstract  Two series of new aromatic polyimides were synthesized from 2,5-bis(4-aminophenyl)-1,3,4-oxadiazole and 2,5-diamino-pyridine and various diamidedianhydrides and dietherdianhydride via high-temperature polycondensation in N-methyl-2-pyrrolidinone. The structures of polymers were characterized by means of FTIR, (1)H NMR spectroscopy, and elemental analysis; the results showed an agreement with the proposed structure. The polymers exhibited high glass-transition temperatures (T(g) = 199-290 degrees C) and high thermal stability with decomposition temperatures (T(d)) in the range of 437-510 degrees C. The optical properties, that is, absorption and photoluminescence (PL) of synthesized polymers, were investigated in solution and in solid state as a blend with inert poly(methyl methacrylate) (PMMA). The polymers in solution emitted violet-blue light in the range of 417-453 nm depending on the polymer structure. The polymers containing pyridine rings in the backbone emitted light at a longer wavelength than polymers with oxadiazole units. The effect of solvent polarity on polymer emission was found. Additionally, the photoluminescence properties of the polymers before and after protonation with HCl were tested.

Abstract  Complexation between poly(methyl vinyl ether-maleic anhydride) copolymer (Gantrez AN 169) and polyvinylpyrrolidone (PVP K-90D) in aqueous solutions were investigated using a viscometric method and Raman spectroscopy. The composite films with different weight ratios of PVP to Gantrez were prepared in the presence of N-methyl-2-pyrrolidone. The release profiles of diclofenac sodium (DS) from these films were determined and the bioadhesive properties measured. An interpolymer complex was formed through hydrogen bonding between the carbonyl groups of PVP and the hydroxyl groups of Gantrez. The formation of interpolymer hydrogen bonds reduced the interaction of the polymers with water molecules, thus resulting in a lower solubility of the complex in water and a further retarded release of DS from the composite films. The interpolymer complexation was also found to increase the bioadhesive properties of the composite films to a silicone elastomer substrate. The complexation of PVP and the Gantrez copolymer in the composite films was a critical factor affecting the release of DS from the films and the bioadhesive properties of the films.

Abstract  The present study investigated the influence of a covalently grown polythiophene (polyTh)-based adlayer on the N-methyl-2-pyrrolidone (NMP) dispersion of chemically modified multi-walled carbon nanotubes (MWCNTs). Poly(thiophen-3-yl-acetic acid) (PTAA), poly(ethylenedioxythiophene) (PEDOT), and PTAA/PEDOT co-polymer have been oxidatively grown from the surface of functional MWCNTs afforded corresponding polyTh(A)-, polyTh(B)-, and polyTh(A/B)-MWCNTs, respectively. To study and quantify the MWCNT particle sedimentation behavior of corresponding dispersion systems, an analytical centrifuge system was used. Comparison of dispersion quality of such polyTh-MWCNT composites with both commercial MWCNTs (COM-MWCNTs) and oxidized MWCNTs (polyCOOH-MWCNTs) was established. Analysis of sedimentation results combined with TGA, Elemental Analysis (EA), and Kaiser Test data showed that the COOH groups present on the surface of MWCNTs strongly promote the dispersability much beyond current sedimentation rate observed for COM-MWCNTs, - sedimentation rates were higher than 80% and below 2% for oxidized polyCOOH-MWCNTs. However, regarding MWCNTs coated with polyTh polymers in a core (MWCNT)-shell (polyTh) structure, outer functional groups are not the only influential factor for promoting high dispersion qualities. For these dual phase composite systems, the weight and thickness of polyTh-shell on the composite must be considered.

Abstract  Pregnant rats (Mol:WIST) were exposed to 150 ppm N-methylpyrrolidone for 6 hours per day on gestation days 7-20. The dose level was selected so as not to induce maternal toxicity or decrease viability of offspring. In the preweaning period, the exposed offspring had a lower body weight and their physical development was delayed. Neurobehavioral evaluation of the male pups revealed no effects on basal functions of the central nervous system. The animals appeared normal and motor function (rotarod), activity level (open field), and performance in learning tasks with a low grade of complexity were similar in the two groups. However, in more difficult tasks such as the reversal procedure in Morris water maze and operant delayed spatial alternation (Skinner boxes), performance was impaired in exposed offspring.

Abstract  Pretargeting with bivalent effectors capable of bridging antitumor antibodies has been reported to provide superior results by affinity enhancement. Morpholinos (MORFs) and other DNA analogues used for pretargeting are ideally suited as bivalent effectors since they are easily synthesized and the distance between binding regions, likely to be a determinant of binding, may be adjusted simply by lengthening the chain. The goal of this investigation was to synthesize a bivalent MORF and to determine by surface plasmon resonance (SPR) whether the bivalent MORF exhibited bimolecular binding and whether the MORFs showed improved in vitro hybridization affinity in its bivalent form compared to its monovalent form. An 18 mer amino-derivitized MORF was made bivalent by dimerizing with disuccinimidyl suberate (DSS) in 1-methyl-2-pyrrolidinone (NMP) with N,N-diisopropylethylamine (DIEA) followed by purification by ion exchange chromatography. The in vitro hybridization affinity of bivalent compared to monovalent MORF was then measured by SPR. For these measurements, the complementary biotinylated cDNA was immobilized at coating densities that provided an average spacing of 20-100 angstroms and used to investigate the influence of this spacing on binding of the bivalent MORF with its binding regions separated by 25 A. The yield of bivalent MORF was as high as 45%, and the structure was confirmed by MALDI-TOF mass spectroscopy. When the sensograms obtained by SPR were analyzed using different binding models, the evidence was consistent with bimolecular binding of the bivalent MORF. The dissociation rate constant of the bivalent compared to monovalent MORF was more than 10-fold lower at 2.14 compared to 0.27 x 10(-5) (1/s) (p < 0.05), and since the association rate constants were similar at 8.53 and 5.64 x 10(5) (1/M.s) (p = 0.08), the equilibrium constant for hybridization to the immobilized cDNA of the bivalent compared to the monovalent MORF was almost 20-fold higher at 3.99 compared to 0.21 x 10(10) (1/M) (p < 0.05). In addition, qualitative evidence for bivalent binding of the bivalent MORF was apparent in the lower concentrations necessary to saturate the cDNA. Finally, the stoichiometry interpretation of the binding data provided estimates of the fraction of bivalent MORF binding bimolecularly. Under one set of conditions, this value was 20%. In conclusion, a bivalent MORF was easily synthesized by dimerization of a monovalent MORF. A lower dissociation rate constant and higher equilibrium constant was measured by SPR for the bivalent compared to monovalent MORF in their binding to an immobilized cDNA. These results show that bimolecular binding was occurring in the case of the bivalent MORF and suggest that bivalency may be superior to monovalency in MORF pretargeting applications.

Abstract  Chitin dissolved in N,N-dimethylacetamide, N-methyl-2-pyrrolidone and their mixed solvents in the presence of 5% LiCl was treated with acetic anhydride-pyridine, and the mixture solution was heated at 100 degrees C for 6 h to give a partially O-acetylated chitin gel. Chitin dissolved in these solvents in the presence of 5% LiCl was mixed with pyridine, and the mixture solution was heated at 60 degrees C for 5 h to give a chitin gel. Both the gels were rigid and transparent, and their properties and the rate of the hydrolysis of the chitin xerogel by hen-egg white lysozyme were essentially similar to those of N-acetylchitosan gel prepared by chemical N-acetylation of chitosan.

Abstract  The objective of this study was to investigate the effect of vehicle systems, pH and enhancers on the permeation of a highly lipophilic basic drug aripiprazole (ARPZ) through human cadaver skin. Solubility of ARPZ in single, binary, tertiary, and quaternary vehicle systems of N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), water, ethanol and isopropyl myristate (IPM) was studied. Gel formulations of 5% ARPZ were developed with 0.5% Carbopol 971P in quaternary vehicle systems consisting of NMP, DMSO, water and ethanol or IPM at optimum ratio of 40/40/5/15. The effect of pH of the gel formulations and fatty acids with different chain lengths on the permeation was studied. The flux of ARPZ from gel formulation with IPM and ethanol was comparable. A four fold increase in APRZ flux was observed when the pH of the gel systems was lowered from pH 8.2 to pH 6 or pH 7. For fatty acids, the order of flux is lauric acid > myristic acid > caprylic acid > oleic acid. In all the cases, in vitro permeation rate of ARPZ through human cadaver skin followed zero order kinetics. This study demonstrated that ARPZ in tertiary vehicle system of NMP/DMSO/water/IPM at ratio of 40/40/5/15 and gel system of Carbopol 971P with pH 7 is a promising candidate for transdermal delivery.

Abstract  A series of Fe(III) salts and organic solvents have been screened to develop novel non-aqueous catalysts for the conversion of H2S to sulfur. FeCl3/95% N-methylpyrrolidinone/5% H2O was found to be a most efficient non-aqueous system. The process chemistry, the proposed mechanism of catalytic oxidation of H2S, and the electrochemistry are discussed.

Abstract  Oxidations of alkanes, alkenes, and aromatic rings with pyridine N-oxides are efficiently catalyzed by ruthenium porphyrins under mild conditions. We show here that the oxidation of N-acyl cyclic amines with RuIVtetraarylporphyrin dichloride-2,6-substituted pyridine N-oxides directly gives N-acyl amino acids in modest to good yield via oxidative C-N bond cleavage. N-Acylpyrrolidines and N-acylpiperidines were converted to N-acyl-gamma-aminobutyric acids and N-acyl-delta-aminovaleric acids, respectively. This type of reaction is a novel one in which the C-N bond is cleaved selectively at the less substituted carbon. Notably, the proline residue in proline-containing peptides was selectively converted to glutamate. A large intramolecular kinetic isotope effect (kH/kD = 9.8) was observed in the oxidation of N-benzoyl[2,2,-d2]pyrrolidine, indicating that the reaction should involve an alpha-hydrogen atom abstraction process as the rate-determining step. N-Acylcarbaldehyde, the putative intermediate ring-opened form of alpha-hydroxylated N-acyl cyclic amine, was readily oxidized with the oxidizing system to afford the corresponding N-acylamino acid in good yield. Further, lactams (1-methyl-2-pyrrolidone and 1-methyl- 2-piperidone) were also oxidized to give the corresponding imides (1-methylsuccinimide and 1-methylpiperidine-2,6-dione).

Abstract  Poor solubility and bioavailability are limiting factors for the clinical application of curcumin. The objective of the current study was to develop a liquid formulation with increased solubility and systemic bioavailability. A co-solvent formulation with increased solubility of 20 mg/ml was developed and optimized. Pharmacokinetics of the new formulation were evaluated using rats receiving 30 mg/kg intravenous or 50 mg/kg intramuscular administration of co-solvent formulation, compared against a control group receiving 50 mg/kg of curcumin in DMSO through intramuscular injection. Plasma concentrations were measured using liquid chromatography-mass spectrometry (LC-MS/MS). The intramuscular injection of formulation resulted in 30% absolute bioavailability and provided sustained release by maintaining plasma concentrations of curcumin above 240 ng/ml for up to 4 h. A 29-fold increase in the maximum plasma concentration (Cmax) and 28-fold increase in the area under the plasma concentration versus time curve (AUC) led to a 28-fold increase in relative bioavailability for the co-solvent formulation. The findings reported here suggest that the clinical application of curcumin can be better-exploited through an intramuscular administration of the co-solvent formulation developed in the present study.

Abstract  Guided bone regeneration (GBR) has been utilized for several decades for the healing of cranio-maxillofacial bone defects and, particularly in the dental field, by creating space with a barrier membrane to exclude soft tissue and encourage bone growth in the membrane-protected volume. Although the first membranes were non-resorbable, a new generation of GBR membranes aims to biodegrade and provide bioactivity for better overall results. The Inion GTR™ poly(lactide-co-glycolide) (PLGA) membrane is not only resorbable but also bioactive, since it includes N-methylpyrrolidone (NMP), which has been shown to promote bone regeneration. In this study, the effects of loading different amounts of NMP onto the membrane through chemical vapour deposition or dipping have been explored. In vitro release demonstrated that lower levels of NMP led to lower NMP concentrations and slower release, based on total NMP loaded in the membrane. The dipped membrane released almost all of the NMP within 15 min, leading to a high NMP concentration. For the in vivo studies in rabbits, 6 mm calvarial defects were created and left untreated or covered with an ePTFE membrane or PLGA membranes dipped in, or preloaded with, NMP. Evaluation of the bony regeneration revealed that the barrier membranes improved bony healing and that a decrease in NMP content improved the performance. Overall, we have demonstrated the potential of these PLGA membranes with a more favourable NMP release profile and the significance of exploring the effect of NMP on these PLGA membranes with regard to bone ingrowth. Copyright © 2014 John Wiley & Sons, Ltd.

Abstract  1-Methyl-2-pyrrolidone (NMP) seeded with 5% trifluoroacetic acid is identified as a singular buffer, polar enough to produce fine electrospray drops, yet having excellent solubility for many industrial polymers such as polystyrene (PSR) and poly(methyl methacrylate) (PMMA). Four PSR mass standards (M = 9.2, 34.5, 68, and 170 kDa) with narrow mass distributions are electrosprayed from their solutions in this buffer. The high charge on the resulting ions is reduced to unity with a radioactive source, whereby their electrical mobility distributions, determined by a differential mobility analyzer, yield unambiguously their size distribution. Each standard produces (at high solution concentration) several mobility peaks associated with the formation of particles containing from one to six polymer molecules, used to establish a relation Z(M) between electrical mobility Z and polymer mass. Within the indeterminacy given by inaccuracies in the nominal masses of the standards, this relation indicates that the polymers form spherical balls with a density close to the bulk density of polystyrene, as seen previously with poly(ethylene glycol) chains. Good mobility spectra from the same buffer are also obtained for PMMA (M = 49 kDa). Because NMP is less conductive and contains more involatile impurities than common aqueous buffers, the electrospray ions formed tend to carry a small contaminant crust, which distorts the inferred mass distribution unless a high spray quality is achieved.

Abstract  The objective was to develop chitosan atorvastatin (ATR) nanocrystals loaded into Poly (lactic-co-glycolic) acid (PLGA) injectable in situ gel (ISG) system that can minimize initial drug burst and enhance hypolipidemic effect. ATR nanocrystals were successfully characterized for size, morphology, crystallinity and drug-excipients interaction. The effects of varied polymer concentration and gelling solvents were evaluated for initial burst release and in vivo efficacy. Short term stability study was also conducted for the promising formulation. Nanocrystals of size 254 nm were prepared using low molecular weight chitosan and were of smooth surface with multiple scaffolds like structures. X-ray powder diffraction revealed the crystalline structure of the prepared nanocrystals while no drug-excipients interactions were observed. Addition of nanocrystals did not significantly alter gelation property of the ISG system that showed acceptable syringeability. The promising ISG formulation was achieved with 45% PLGA in N-methyl pyrrolidone: benzyl benzoate (1:3). In-vitro dissolution study illustrated lower initial ATR burst and prolonged drug release from nanocrystal based ISG when compared to plain ATR ISG. The pharmacokinetic and hypolipidemic biochemical parameters were comparable in the two formulations. The promising formulation exhibited minimum drug degradation at 4 &#176;C and so could be considered as an ideal ISG delivery system.

Abstract  To investigate the utilities of a shed snake skin as a model membrane for preclinical studies of transdermal drug delivery, the flux of indomethacin was determined under various conditions by using a diffusion cell. The flux of fatty alcohols was determined and compared with that in human skin reported in references. The esterase activity of shed snake skin was also determined. It was found that the flux of indomethacin decreased with an increase of pH and the amount of ethanol in a vehicle. The flux of indomethacin increased by the addition of Azone, N-methyl-2-pyrrolidone and N,N-dimethyl-m-toluamide in the cream. The flux of fatty alcohols in shed snake skin was greater than that reported in human skin, and shed snake skin had similar esterase activity to human skin.

Abstract  Epoxyols are generally accepted as crucial intermediates in lipid oxidation. The reactivity of tert-butyl (9R,10S,11E,13S)-9, 10-epoxy-13-hydroxy-11-octadecenoate (11a,b) toward lysine moieties is investigated, employing N(2)-acetyllysine 4-methylcoumar-7-ylamide (12) as a model for protein-bound lysine. The prefixes R and S denote the relative configuration at the respective stereogenic centers. Independent synthesis and unequivocal structural characterization are reported for 11a,b, its precursors, and tert-butyl (9R,10R,11E, 13S)-10-(¿5-(acetylamino)-6-[(4-methyl-2-oxo-2H-chromen-7-yl)amino ]-6 -oxohexyl¿amino)-9,13-dihydroxy-11-octadecenoate (13a-d). Reactions of 11a,b and 12 in 1-methyl-2-pyrrolidone (MP) and MP/water mixtures at pH 7.4 and 37 degrees C for 56 days show formation of the aminols 13a-d to be favored by an increased water content. The same trend is observed for hydrolytic cleavage of 11a,b to tert-butyl (E)-9,10, 13-trihydroxy-11-octadecenoate (14) and tert-butyl (E)-9,12, 13-trihydroxy-10-octadecenoate (15). Under the given conditions, aminolysis proceeds via an S(N)2 substitution, in contrast with the S(N)1 process for hydrolysis. In the MP/water (8:2) incubation, 15. 8% of 12 has been transformed to 13a-d and 10.5% of 11a,b hydrolyzed to the regioisomers 14 and 15 after 8 weeks, respectively. Aminolysis of alpha,beta-unsaturated epoxides by lysine moieties therefore is expected to be an important mode of interaction between proteins and lipid oxidation products.

Abstract  PURPOSE: Microemulsion (ME) systems allow for the microscopic co-incorporation of aqueous and organic phase liquids. In this study, the phase diagrams of four novel ME systems were characterized.

METHODS: Water and IPM composed the aqueous and organic phases respectively, whereas Tween 80 served as a nonionic surfactant. Transdermal enhancers such as n-methyl pyrrolidone (NMP) and oleyl alcohol were incorporated into all systems without disruption of the stable emulsion.

RESULTS: A comparison of a W/O ME with an O/W ME of the same system for lidocaine delivery indicated that the O/W ME provides significantly greater flux (p < 0.025). The water phase was found to be a crucial component for flux of hydrophobic drugs (lidocaine free base, estradiol) as well as hydrophilic drugs (lidocaine HCl, diltiazem HCl). Furthermore, the simultaneous delivery of both a hydrophilic drug and a hydrophobic drug from the ME system is indistinguishable from either drug alone. Enhancement of drug permeability from the O/W ME system was 17-fold for lidocaine free base, 30-fold for lidocaine HCl, 58-fold for estradiol, and 520-fold for diltiazem HCl.

CONCLUSIONS: The novel microemulsion systems in this study potentially offers many beneficial characteristics for transdermal drug delivery.

Abstract  In this study, uniform nitrogen-doped carbon quantum dots (N-CDs) were synthesized through a one-step solvothermal process of cyclic and nitrogen-rich solvents, such as N-methyl-2-pyrrolidone (NMP) and dimethyl-imidazolidinone (DMEU), under mild conditions. The products exhibited strong light blue fluorescence, good cell permeability and low cytotoxicity. Moreover, after a facile post-thermal treatment, it developed a lotus seedpod surface-like structure of seed-like N-CDs decorating on the surface of carbon layers with a high proportion of quaternary nitrogen moieties that exhibited excellent electrocatalytic activity and long-term durability towards the oxygen reduction reaction (ORR). The peak potential was -160 mV, which was comparable to or even lower than commercial Pt/C catalysts. Therefore, this study provides an alternative facile approach to the synthesis of versatile carbon quantum dots (CDs) with widespread commercial application prospects, not only as bioimaging probes but also as promising electrocatalysts for the metal-free ORR.

Abstract  The aim of this study was to explore the use of Pharmasolve as a new kind of permeability enhancer in ophthalmic drug delivery systems. The ocular irritation of different concentrations of Pharmasolve on rabbit eyes was evaluated in detail. Four drugs ranging from hydrophilic to lipophilic, namely ribavirin, puerarin, enoxacin, and ibuprofen, were used as model compounds to investigate the effects of different concentrations of Pharmasolve on the corneal permeability. The mechanism of ocular permeation enhancement of drugs by Pharmasolve was also discussed. The results showed that Pharmasolve presented no irritation when the concentration used was lower than 10%. Pharmasolve could enhance the ocular permeability of the four test drugs; the maximum enhancement in P(app) was 4.04, 2.76, and 2.67-fold for ribavirin, enoxacin, and puerarin, respectively; 2.5% (v/v) Pharmasovle increased the P(app) by about 1.47-fold for ibuprofen; which suggested that it would have a great potential to be used as a safe and effective penetration enhancer in ocular drug delivery systems in the future.

Abstract  The involvement of cytochrome P450 2E1 (CYP2E1) in the metabolism of N-methyl-2-pyrrolidone (NMP) was studied with three experimental approaches: in the rat, in vitro in human microsomes, and in human volunteers. NMP was administered dermally (40 mg/kg) to OFA rats to examine the influence of CYP2E1 inhibition (5 mg/kg diethyldithiocarbamate, DETC, 30 min before) and CYP2E1 induction (after 4 days of fasting). The main NMP metabolite 5-hydroxy- N-methylpyrrolidone (5HNMP) in the urine fractions collected during the following 48 h was analysed by gas chromatography-mass spectrometry. CYP2E1 inhibition led to a statistically significant retardation of 5HNMP excretion in urinary fractions collected during the first 12 h. In the group of fasted rats, a two-fold increase of CYP2E1 activity was observed in comparison with the control group. During the first 6 h after dermal administration of NMP to fasted rats, about 33% of the dose was excreted in urine versus 22% in controls. In vitro, NMP (15 mM) was incubated (up to120 min) with human liver microsomes and the formation of 5HNMP followed Michaelis-Menten kinetics with V(max) of 1.1 nmol/min per mg protein and K(m) of 2.4 mM. The formation of 5HNMP was inhibited by 35% in the presence of a monoclonal antibody against CYP2E1, but not by CYP1A2 antibody. In a dermal application experiment, 12 humans volunteers were exposed by means of a dermal patch to 300 mg NMP; five urine fractions were collected during the 48 h following the onset of application in order to measure the major metabolites 5HNMP and 2-hydroxymethylsuccinimide (2HMSI). Before NMP application, a blood sample was collected for the quantification of CYP2E1 mRNA in peripheral blood lymphocytes (PBLs). The mean dermal absorption of NMP was 67.9%. The highest amount of 5HNMP was excreted in urine in the fraction collected between 6-12 h (12.6% of dose), while 2HMSI peaked in fractions 12-24 h and 36-48 h (3.3 and 3.2% of dose, respectively). A significant relationship was found between CYP2E1 mRNA content in PBLs and the amount of both the metabolites excreted in urine within 24 h ( r(2)=0.54, P<0.01). It is concluded that CYP2E1 is involved in the first steps of NMP metabolism in the rat and, to a lesser extent, in humans. Since large variations in CYP2E1 activity exist in the human population (at least 5-fold range), it seems justified to take into account the activity of this enzyme in an individual for an accurate interpretation of biological monitoring of exposure to NMP when relying on 5HNMP and/or 2HMSI determination in urine.