Pharmacokinetics studies of eugenol in Pacific white shrimp (Litopenaeus vannamei) after immersion bath.

BACKGROUND: Eugenol is the most commonly used plant anesthetic to relieve the stressors during various aquaculture procedures. This study aims to investigate the pharmacokinetics of eugenol in Pacific white shrimp by immersion baths in a simulated transportation. RESULTS: The pharmacokinetics of eugenol were firstly investigated in Pacific white shrimp by immersion baths of 300 mg L- 1 eugenol over 5 min (Treatment 1), 10 mg L- 1 eugenol during 24 h (Treatment 2) and a sequential immersion administration (Treatment 3). Concentrations of eugenol in hemolymph, hepatopancreas, and muscle were determined using Gas chromatography-tandem mass spectrometry (GC-MS/MS). After immersion bath of Treatment 1, the elimination half-life (t1/2z) values are 1.3 h and 11 h for hepatopancreas and muscles, indicating the rapid absorption and elimination of eugenol in shrimp. Under the Treatment 2 administration, the eugenol peak concentration is 6527.9 µg/kg in muscle, followed by 402.8 µg/kg in hepatopancreas, with the lowest concentration of 37.9 µg/L in hemolymph. Area under the curve (AUC0-∞) values lie in the order of muscle > hepatopancreas > hemolymph, suggesting that eugenol tends to accumulate in muscle by the immersion administration. Moreover, the average residence time (MRT0-∞) values of 38.6, 23.0 and 115.3 h for hemolymph, hepatopancreas and muscle are achieved, which may indicate that hepatopancreas is the main organ for elimination of eugenol. After combining the conditions in a sequential bath immersion of eugenol (Treatment 3), the maximum concentration (Cmax) values of eugenol are higher than those achieved in Treatment 2, indicating that accumulation of eugenol happened in haemolymph, hepatopancreas and muscle. In addition, the corresponding t1/2z values are 4.7, 14.9 and 47.6 h, respectively, suggesting the faster elimination from the tissues following sequential administration. After the immersion bath, eugenol concentrations in muscle of Pacific white shrimp are lower than 2.5 mg/kg at 2 h, 48 h and 24.5 h in Treatment 1 ~ 3. CONCLUSIONS: A withdrawal period of 2 h, 48 h and 24.5 h following a 300 mg L- 1 of eugenol over a 5-min, 10 mg L- 1 eugenol concentration during a 24-h and combined conditions in a sequential immersion bath were suggested.

Pharmacokinetic Study of Safrole and Methyl Eugenol after Oral Administration of the Essential Oil Extracts of Asarum in Rats by GC-MS.

Asarum is a traditional medicine and has been widely used as remedies for inflammatory diseases, toothache, headache, local anesthesia, and aphthous stomatitis in China, Japan, and Korea. Our previous research found that safrole and methyl eugenol were vital compounds that contribute to distinguish the different species and raw Asarum and its processed products apart. The pharmacokinetics of safrole and methyl eugenol after oral administration of Asarum extract has not been reported yet. In this study, a rapid and simple gas chromatography-mass spectroscopy (GC-MS) method that has a complete run time of only 4.5 min was developed and validated for the simultaneous determination and pharmacokinetic study of safrole and methyl eugenol in rat plasma after administration of Asarum extracts. The chromatographic separation was realized on a DB-17 column (30 m × 0.25 mm × 0.25 µm). And detection was carried out under selected ion monitoring (SIM) mode. Plasma samples were pretreated by n-hexane. The pharmacokinetic parameters provided by this study will be beneficial for further developments and clinical applications of Asarum.

Evaluation of dietary dose administration as an alternative to oral gavage in the rodent uterotrophic and Hershberger assays.

The rodent uterotrophic and Hershberger assays evaluate potential estrogenic and (anti)-androgenic effects, respectively. Both US EPA and OECD guidelines specify that test substance is administered daily either by subcutaneous injection or oral gavage. However, dietary administration is a relevant exposure route for agrochemical regulatory toxicology studies due to potential human intake via crop residues. In this study, equivalent doses of positive control chemicals administered via dietary and gavage routes of administration were compared in the uterotrophic (17α-ethinyl estradiol) and Hershberger (flutamide, linuron, dichloro-2,2-bis(4-chlorophenyl) ethane; 4,4'-DDE) assays in ovariectomized and castrated rats, respectively. For all positive control chemicals tested, statistically significant changes in organ weights and decreases in food consumption were observed by both routes of test substance administration. Decreased body weight gain observed for dietary linuron and 4,4'-DDE indicated that the maximum tolerated dose was exceeded. Hershberger dietary administration resulted in a similar blood exposure (AUC24) for each positive control chemical when compared to gavage. Overall, the correlation in organ weight changes for both the uterotrophic and Hershberger assays suggest that dietary administration is an acceptable route of exposure with similar sensitivity to oral gavage dosing for evaluation of the endocrine potential of a test substance and represents a more appropriate route of test substance administration for most environmental exposure scenarios.

Antinociceptive Interaction and Pharmacokinetics of the Combination Treatments of Methyleugenol Plus Diclofenac or Ketorolac.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the main types of drugs used to treat pain, they have several adverse effects, and such effects can be reduced by combining two analgesic drugs. The aim of this study was to evaluate the nociceptive activity of methyleugenol combined with either diclofenac or ketorolac, and determine certain parameters of pharmacokinetics. For the isobolographic analysis, the experimental effective dose 30 (ED30) was calculated for the drugs applied individually. With these effective doses, the peak plasma concentration (Cmax) was found and the other parameters of pharmacokinetics were established. Methyleugenol plus diclofenac and methyleugenol plus ketorolac decreased licking behavior in a dose-dependent manner in phase II, with an efficacy of 32.9 ± 9.3 and 39.8 ± 9.6%, respectively. According to the isobolographic analysis, the experimental and theoretical ED30 values were similar for methyleugenol plus diclofenac, suggesting an additive effect, but significantly different for methyleugenol plus ketorolac (3.6 ± 0.5 vs. 7.7 ± 0.6 mg/kg, respectively), indicating a probable synergistic interaction. Regarding pharmacokinetics, the only parameter showing a significant difference was Cmax for the methyleugenol plus diclofenac combination. Even with this difference, the combinations studied may be advantageous for treating inflammatory pain, especially for the combination methyleugenol plus ketorolac.

Self-Micro-Emulsifying Controlled Release of Eugenol Pellets: Preparation, In vitro/In vivo Investigation in Beagle Dogs.

This report aimed to formulate self-micro-emulsifying (SMEDDS) controlled-release pellets delivery system to improve aqueous solubility and in vivo availability of eugenol, a main constituent of clove oil with multiple pharmacological activities. The optimal formulation of eugenol-SMEDDS was eugenol: ethyl oleate: cremophor EL: 1, 2-propylene glycol at the ratio of 5:5:12:8. The SMEDDS were observed under transmission electron microscopy (TEM), and the size distribution was measured with dynamic laser light scatting (DLS). The particle size, index of dispersity (PDI), and zeta potential (Z-potential) were 68.8 ± 0.1 nm, 0.285 ± 0.031, and - 11.62 ± 0.63 mV, respectively. Eugenol-SMEDDS exhibited substantial increased in vitro dissolution compared with the free eugenol. The eugenol-SMEDDS sustained-release pellets (eugenol-SMEDDS-SR pellets) comprising of eugenol-SMEDDS, hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), and ethyl cellulose (EC) coats were obtained via extrusion spheronization technique. Consequently, the obtained pellets observed under scanning electron microscopy (SEM) showed spherical shape with smooth surface, desirable drug loading capacity (7.18 ± 0.17%), greater stability, and controlled release. Meanwhile, the oral test showed that bioavailability of eugenol in pellets was highly improved 23.6-fold to the free eugenol. Overall, these results suggested that the improvement of the oral bioavailability of eugenol-SMEDDS-SR could be due to the successful incorporation of the drug into SMEDDS.

Hybrid Ofloxacin/eugenol co-loaded solid lipid nanoparticles with enhanced and targetable antimicrobial properties.

In the global context of an imminent emergence of multidrug-resistant microorganisms, the present work combined the use of nanotechnology and the therapeutic benefits of natural compounds as a strategy to potentiate antimicrobial action of the wide-spectrum antibiotic Ofloxacin (Ofx). Hybrid solid lipid nanoparticles (SLN) were synthesized by incorporation of chitosan (Chi, a cationic biopolymer with antimicrobial activity) and eugenol (Eu, a phenolic compound that interferes with bacterial quorum sensing) into a lipid matrix by hot homogenization/ultrasonication method. The developed SLN/Chi/Eu sustainably released the encapsulated Ofx for 24 h. Characterization by DLS, TEM, DSC, TGA and XRD revealed the presence of positively charged spherical nanoparticles with diameters around 300 nm and Ofx entrapped in amorphous state. The SLN exhibited an enhanced bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) for free and nanoencapsulated Ofx formulations was below 1.0 µg/ml. The MIC values decreased by 6.1- to 16.1-fold when Ofx was encapsulated in SLN/Chi/Eu. Fluorescent-labeled nanoparticles had the ability to interact with the bacterial cell membrane. Selective toxicity of SLN/Chi/Eu-Ofx was tested in the range of 0.3-30.0 µg/ml and showed no toxicity up to 3.0 µg/ml Ofx in human cell models (A549 and Wi-38) at 24 h and 48 h exposure. It was proved that the administration of hybrid SLN to mice by dry powder inhalation reached therapeutic Ofx levels in lungs.

Quantification and Brain Targeting of Eugenol-Loaded Surface Modified Nanoparticles Through Intranasal Route in the Treatment of Cerebral Ischemia.

OBJECTIVE: To enhance brain bioavailability for intranasally administered Eugenol-encapsulated-chitosan-coated-PCL-Nanoparticles (CS-EUG-PCL-NPs). METHODS: Chitosan-coated-PCL-Nanoparticles (CS-PCL-NPs) were developed through double emulsification-solvent evaporation technique and further characterized for particle size, zeta potential, size distribution, encapsulation efficiency as well as in vitro drug release. UPLC-PDA method was developed to evaluate brain-drug uptake for optimized CS-EUG-PCL-NPs and to determine it's pharmacokinetic in rat's brain as well as plasma. RESULTS: Mean particles size (224.5±5.31), polydispersity index (PDI) i. e. (0.216±0.020) and entrapment efficiency (68.13±5.03) was determined for developed NPs. UPLC-PDA-eλ study showed a significantly high mucoadhesive potential of CS-EUG-PCL-NPs and least for conventional and homogenized nanoformulation; elution time for EUG and internal standard (IS) thymoquinone as 3.50 and 3.61 min were observed respectively. Furthermore, intra and inter-assay (%CV) of 0.25-1.57, %accuracy (97.11-99.00%) as well as a linear dynamic range (100.00 ng/mL-2500.0 ng/mL), was observed. Pharmacokinetic studies in Wistar rat brain and plasma exhibited a high AUC0-24 alongwith an amplified Cmax (p**<0.01) as compared to i. v. treated group. CONCLUSIONS: Intranasal administration of developed CS-coated-EUG-loaded-PCL-NPs enhanced the drug bioavailability in rat brain and thus preparation of Eugenol-NPs may help treat cerebral ischemia effectively. The toxicity studies performed at the end revealed safe nature of optimized nanoformulation.

Eugenol derivatives prospectively inhibit l-asparaginase: A heady target protein of Salmonella typhimurium.

Salmonella typhimurium is the causative agent of severe human infections and mortality throughout the world. Pacing advent of new resistance mechanisms in this microorganism exists, rendering treatment of infectious disease difficult. Ciprofloxacin is no longer considered the first choice of antimicrobial agent due to the emergence of resistance. Therefore, the need for scenario is to find out novel drug target and its potential inhibitor to fight against this pathogen. The present study was undertaken to find out a novel drug target and its inhibitor for improving the current therapeutic methods for treating Salmonella infections. It is found that l-asparaginase is exploited by the pathogen for its survival benefit. Therefore, it could be targeted to fight against lethality caused by Salmonella infections. In the present in silico study, the 3-D structure of the enzyme l-asparaginase was modelled by using homology modeling technique. Thereafter, molecular docking studies and ADMET prediction to assess pharmacokinetic profiles of test ligands (eugenol and its derivative) was performed. The results show that eugenol and its derivative are capable of inhibiting the Salmonella virulent protein l-asparaginase. There were 18 ligands including ciprofloxacin (used as reference) were docked. The lowest binding energy was observed with eugenol derivative 8 i.e -5.836 kcal/mol while for ciprofloxacin was -4.661 kcal/mol. The docking of the eugenol derivative 8 with l-asparaginase revealed a strong interaction between them with two hydrogen bonds. Thr 35 and Asp 116 residues are actively participating in this interaction. The result of ADMET profiling suggests the potency of eugenol and its derivatives against Salmonellal-asparaginase-II as a compelling drug candidate. These findings provide useful information on the biological role, structure-based drug design and potent inhibitor of l-asparaginase for the development of effective therapeutic molecule against Salmonella infection.

Elimination kinetics of eugenol in grass carp in a simulated transportation setting.

BACKGROUND: Fish are vulnerable to stress from over-crowding during transportation and eugenol is the most common sedative used to minimize fish injury. The ADI value of 2.5 mg/kg is recommended by the Joint FAO/WHO Expert Committee on Food Additives. The aim of this work was to study the elimination kinetics of eugenol following exposure of grass carp to a eugenol bath in a simulated transportation setting. RESULTS: Grass carp, Ctenopharyngodon idella (120 fish) were exposed for 24 h to a 10 mg/L eugenol bath. Sampling was performed during a 96 h period after the 24 h bath. Eight fish were sampled at each time point and muscle, plasma and liver concentrations of the drug were determined by ultra-performance liquid chromatography-tandem mass spectrometry. The concentration-time data of eugenol in each tissue were analyzed using non-compartmental methods. The peak concentrations (Cmax) in plasma, muscle and liver were 7.68, 5.30 and 24.63 mg/kg and the elimination half-lives (t1/2ß) were 19.79, 10.27 and 55.28 h, respectively. The clearance (CL) values were 0.10, 0.44 and 0.04 L/h/kg and the areas under the concentration-time curve (AUC0-96h) were 91.54, 22.44, and 214.12 mg·h/L in plasma, muscle and liver, respectively. After a eugenol exposure bath, drug concentrations in muscle tissue of grass carp were below 1 mg/kg at 8 h and 0.1 mg/kg at 24 h. CONCLUSIONS: The drug concentrations in muscle tissue at 8 h were lower than the recommended ADI value.

[Transdermal permeation of effective components in Huoxue Zhitong patch].

To establish a determination method for the contents of paeonol, eugenol and piperine in receptor liquid and to research the transdermal permeability of Huoxue Zhitong patch. The contents of paeonol, eugenol and piperine in receptor liquid were determined by high pressure liquid chromatography(HPLC); and the receptor liquid was optimized by taking accumulative amount penetrated within 24 hours, percutaneous permeation rate and skin irritation as indexes. In vitro Franz diffusion experiment was applied to assess the percutaneous penetration characteristics and regularity of Huoxue Zhitong patch. The results showed that the accumulative penetration amount and penetration rate by using PEG 400-ethanol-normal saline 3∶3∶4 as receptor liquid were higher than those by using propylene glycol∶ethanol∶normal saline 3∶3∶4 and ethanol-normal saline 3∶7, the and skin irritation of PEG 400-ethanol-normal saline 3∶3∶4 was smaller than propylene glycol:ethanol: normal saline 3∶3∶4. Results of percutaneous permeability experiments displayed that the accumulative amount penetrated of paeonol, eugenol and piperine within 24 hours was 2.84, 19.9, and 0.753 µg•cm⁻² respectively in Huoxue Zhitong patch and the penetration rate was 0.18, 1.22, and 0.02 µg•cm⁻²â€¢h⁻¹ respectively. Thus, the permeation of paeonol, eugenol and piperine through the skin was a diffusion process, which was irrelevant to their content in patch.

[Determination and pharmacokinetics of main components for Psoralea corylifolia-Myristica fragrants drug pair by using UPLC-MS/MS].

To conduct multiple-reaction monitoring(MRM) quantitative analysis with ultra-high performance liquid chromatography coupled with mass spectrometry method(UPLC-MS/MS), determine the concentrations of psoralen, isopsoralen, bakuchiol and dehydrodiisoeugenol in plasma under positive iron mode with chloramghenicol as internal standard, and investigate the pharmacokinetics process of the main components before and after oral administration of drug pair Psoralea corylifolia -Myristica fragrants. Thirty-six SD rats were randomly divided into three group(A, B, C) and received P. corylifolia extract, P. corylifolia-M. fragrants extract, and M. fragrants extract respectively by intragastric administration. The plasma samples were collected at different time points. In the plasma samples, psoralen, isopsoralen, bakuchiol and dehydrodiisoeugenol showed good linear relationship within concentration rages of 0.098 125 to 39.25, 0.084 37 to 33.75, 0.046 875 to 18.75, and 0.11 to 2.2 mg•L⁻¹ respectively. The precision and stability results showed that the determination method of plasma concentration for such compositions was stable and reliable. The pharmacokinetic parameters obtained by DAS 2.0 showed varying differences before and after compatibility. According to the experimental results, the compatibility of P. corylifolia and M. fragrants can significantly impact the pharmacokinetic process of main components, expand their distribution and accelerate their metabolism and elimination in vivo.

Neuroprotective effects of eugenol against aluminiuminduced toxicity in the rat brain.

Aluminium (Al) is a neurotoxic metal that contributes to the progression of several neurodegenerative diseases. The aim of the present study was to evaluate the protective effect of dietary eugenol supplementation against aluminium (Al)- induced cerebral damage in rats. Male Wistar rats were divided into four groups: normal controls, rats fed a diet containing 6,000 µg g-1 eugenol, rats intoxicated daily with aluminium chloride (84 mg kg-1 body weight) p. o. and fed either a basal diet or a eugenol-containing diet. Daily oral administration of Al for four consecutive weeks to rats significantly reduced brain total antioxidant status (TAS) (11.42±0.31 µmol g-1 tissue, p<0.001) with a subsequent significant enhancement of lipid peroxidation (MDA) (32.55±1.68 nmol g-1 tissue, p<0.002). In addition, Al enhanced brain acetylcholinesterase activity (AChE) (46.22±4.90 U mg-1 protein, p<0.001), tumour necrosis factor alpha (TNF-α) (118.72±11.32 pg mg-1 protein, p<0.001), and caspase 3 (Casp-3) (8.77±1.26 ng mg-1 protein, p<0.001) levels, and in contrast significantly suppressed brain-derived neurotrophic factor (BDNF) (82.74±14.53 pg mg-1 protein, p<0.002) and serotonin (5-HT) (1.54±0.12 ng mg-1 tissue, p<0.01) levels. Furthermore, decreased glial fibrillary acidic protein (GFAP) immunostaining was noticed in the striatum of Al-intoxicated rats, compared with untreated controls. On the other hand, co-administration of dietary eugenol with Al intoxication restored brain BDNF (108.76±2.64 pg mg-1 protein) and 5-HT (2.13±0.27 ng mg-1 tissue) to normal levels, enhanced brain TAS (13.43±0.24 µmol g-1 tissue, p<0.05), with a concomitant significant reduction in TNF-α (69.98±4.74 pg mg-1 protein) and Casp-3 (3.80±0.37 ng mg-1 protein) levels (p<0.001), as well as AChE activity (24.50±3.25 U mg-1 protein, p<0.001), and increased striatal GFAP immunoreactivity, compared with Al-treated rats. Histological findings of brain tissues verified biochemical data. In conclusion, eugenol holds potential as a neuroprotective agent through its hydrophobic, antioxidant, and anti-apoptotic properties, as well as its neurotrophic ability against Al-induced brain toxicity in rats.

Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels.

Methyleugenol is a rodent hepatocarcinogen occurring in many herbs and spices as well as essential oils used for flavoring. Following metabolic activation by cytochromes P450 (CYPs) and sulfotransferases (SULTs), methyleugenol can form DNA adducts. Previously, we showed that DNA adduct formation by methyleugenol in mouse liver is dependent on SULT1A1 expression and that methyleugenol DNA adducts are abundant in human liver specimens. In humans, SULT1A1 activity is affected by genetic polymorphisms, including single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). Here we investigated the relationship between individual methyleugenol DNA adduct levels and SULT1A1 in human liver samples. Using isotope-dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry, we quantified methyleugenol DNA adducts in 121 human surgical liver samples. Frequent CNVs, including deletions (f = 3.3%) and duplications (f = 36.4%) of SULT1A1, were identified using qPCR and TaqMan assays in the donors' genomic DNA. SULT1A1 mRNA and protein levels were quantified using microarray data and Western blot analysis, respectively. Methyleugenol DNA adducts were detected in all 121 liver samples studied. Their levels varied 122-fold between individuals and were significantly correlated to both mRNA and protein levels of SULT1A1 (r s = 0.43, and r s = 0.44, respectively). Univariate and multivariate statistical analysis identified significant associations of SULT1A1 CNVs with mRNA (p = 1.7 × 10-06) and protein (p = 4.4 × 10- 10) levels as well as methyleugenol DNA adduct levels (p = 0.003). These data establish the importance of SULT1A1 genotype for hepatic methyleugenol DNA adducts in humans, and they confirm a strong impact of SULT1A1 CNVs on SULT1A1 hepatic phenotype.

Comprehensive evaluation of carboxylated nanodiamond as a topical drug delivery system.

The best strategy in the development of topical drug delivery systems may be to facilitate the permeation of drugs without any harmful effects, while staying on the skin surface and maintaining stability of the system. Nanodiamonds (NDs) play a key role with their excellent physicochemical properties, including high biocompatibility, physical adsorption, reactive oxygen species (ROS) scavenging capability, and photostabilizing activity. Z-average sizes of carboxylated ND (ND-COOH) agglutinate decreased significantly as the pH increased. Fluorescein-conjugated ND was observed only on the stratum corneum, and no sample diffused into the dermal layer even after 48 hours. Moreover, ND-COOH and ND-COOH/eugenol complex did not show significant toxic effects on murine macrophage cells. ND improved in vitro skin permeation >50% acting as a "drug reservoir" to maintain a high drug concentration in the donor chamber, which was supported by quartz crystal microbalance results. Moreover, ND-COOH could adsorb a drug amount equivalent to 80% of its own weight. A photostability study showed that ND-COOH increased the photostability ~47% with regard to rate constant of the eugenol itself. A significant decrease in ROS was observed in the ND-COOH and ND-COOH/eugenol complex compared with the negative control during intracellular ROS assay. Moreover, ROS and cupric reducing antioxidant capacity evaluation showed that ND-COOH had synergistic effects of antioxidation with eugenol. Therefore, ND-COOH could be used as an excellent topical drug delivery system with improved permeability, higher stability, and minimized safety issue.

Analysis of real-time mixture cytotoxicity data following repeated exposure using BK/TD models.

Cosmetic products generally consist of multiple ingredients. Thus, cosmetic risk assessment has to deal with mixture toxicity on a long-term scale which means it has to be assessed in the context of repeated exposure. Given that animal testing has been banned for cosmetics risk assessment, in vitro assays allowing long-term repeated exposure and adapted for in vitro - in vivo extrapolation need to be developed. However, most in vitro tests only assess short-term effects and consider static endpoints which hinder extrapolation to realistic human exposure scenarios where concentration in target organs is varies over time. Thanks to impedance metrics, real-time cell viability monitoring for repeated exposure has become possible. We recently constructed biokinetic/toxicodynamic models (BK/TD) to analyze such data (Teng et al., 2015) for three hepatotoxic cosmetic ingredients: coumarin, isoeugenol and benzophenone-2. In the present study, we aim to apply these models to analyze the dynamics of mixture impedance data using the concepts of concentration addition and independent action. Metabolic interactions between the mixture components were investigated, characterized and implemented in the models, as they impacted the actual cellular exposure. Indeed, cellular metabolism following mixture exposure induced a quick disappearance of the compounds from the exposure system. We showed that isoeugenol substantially decreased the metabolism of benzophenone-2, reducing the disappearance of this compound and enhancing its in vitro toxicity. Apart from this metabolic interaction, no mixtures showed any interaction, and all binary mixtures were successfully modeled by at least one model based on exposure to the individual compounds.

Prediction of the metabolic clearance of benzophenone-2, and its interaction with isoeugenol and coumarin using cryopreserved human hepatocytes in primary culture.

Benzophenone-2 (BP2) is widely used as a UV screen in both industrial products and cosmetic formulations, where it is frequently found associated with fragrance compounds, such as isoeugenol and coumarin. BP2 is now recognized as an endocrine disruptor, but to date, no information has been reported on its fate in humans. The intrinsic clearance (Clint) and metabolic interactions of BP2 were explored using cryopreserved human hepatocytes in primary cultures. In vitro kinetic experiments were performed to estimate the Michaelis-Menten parameters. The substrate depletion method demonstrated that isoeugenol was cleared more rapidly than BP2 or coumarin (Clint = 259, 94.7 and 0.40 µl/min/10(6) cells respectively). This vitro model was also used to study the metabolic interactions between BP2 and isoeugenol and coumarin. Coumarin exerted no effects on either isoeugenol or BP2 metabolism, because of its independent metabolic pathway (CYP2A6). Isoeugenol appeared to be a potent competitive substrate inhibitor of BP2 metabolism, equivalent to the specific UGT1A1 substrate: estradiol. Despite the fact that inhibition of UGT by xenobiotics is not usually considered to be a major concern, the involvement of UGT1A1 in BP2 metabolism may have pharmacokinetic and pharmacological consequences, due to the its polymorphisms in humans and its pure estrogenic effect.

Utilization of nanotechnology to enhance percutaneous absorption of acyclovir in the treatment of herpes simplex viral infections.

This study aimed to formulate an optimized acyclovir (ACV) nanoemulsion hydrogel in order to provide a solution for the slow, variable, and incomplete oral drug absorption in patient suffering from herpes simplex viral infection. Solubility of ACV in different oils, surfactants, and cosurfactants was explored utilizing a cubic model mixture design to obtain a nanoemulsion with minimum globule size. Preparation of an optimized ACV nanoemulsion hydrogel using a three-factor, three-level Box-Behnken statistical design was conducted. The molecular weight of chitosan (X1), percentage of chitosan (X2), and percentage of Eugenol as a skin permeation enhancer (X3) were selected to study their effects on hydrogel spreadability (Y1) and percent ACV permeated through rat skin after 2.5 hours (Y2). A pharmacokinetic study of the optimized ACV nanoemulsion hydrogel was conducted in rats. Mixtures of clove oil and castor oil (3:1 ratio), Tween 80 and Span 80 (3:1 ratio), and propylene glycol and Myo-6V (3:1 ratio) were selected as the oil, surfactant, and cosurfactant phases, respectively. Statistical analysis indicated that the molecular weight of chitosan has a significant antagonistic effect on spreadability, but has no significant effect on the percent ACV permeated. The percentage of chitosan also has a significant antagonistic effect on the spreadability and percent ACV permeated. On the other hand, the percentage of Eugenol has a significant synergistic effect on percent ACV permeated, with no effect on spreadability. The ex vivo study demonstrated that the optimized ACV nanoemulsion hydrogel showed a twofold and 1.5-fold higher permeation percentage than the control gel and marketed cream, respectively. The relative bioavailability of the optimized ACV nanoemulsion hydrogel improved to 535.2% and 244.6% with respect to the raw ACV hydrogel and marketed cream, respectively, confirming improvement of the relative bioavailability of ACV in the formulated nanoemulsion hydrogel.

BK/TD models for analyzing in vitro impedance data on cytotoxicity.

The ban of animal testing has enhanced the development of new in vitro technologies for cosmetics safety assessment. Impedance metrics is one such technology which enables monitoring of cell viability in real time. However, analyzing real time data requires moving from static to dynamic toxicity assessment. In the present study, we built mechanistic biokinetic/toxicodynamic (BK/TD) models to analyze the time course of cell viability in cytotoxicity assay using impedance. These models account for the fate of the tested compounds during the assay. BK/TD models were applied to analyze HepaRG cell viability, after single (48 h) and repeated (4 weeks) exposures to three hepatotoxic compounds (coumarin, isoeugenol and benzophenone-2). The BK/TD models properly fit the data used for their calibration that was obtained for single or repeated exposure. Only for one out of the three compounds, the models calibrated with a single exposure were able to predict repeated exposure data. We therefore recommend the use of long-term exposure in vitro data in order to adequately account for chronic hepatotoxic effects. The models we propose here are capable of being coupled with human biokinetic models in order to relate dose exposure and human hepatotoxicity.

Evaluation of the interindividual human variation in bioactivation of methyleugenol using physiologically based kinetic modeling and Monte Carlo simulations.

The present study aims at predicting the level of formation of the ultimate carcinogenic metabolite of methyleugenol, 1'-sulfooxymethyleugenol, in the human population by taking variability in key bioactivation and detoxification reactions into account using Monte Carlo simulations. Depending on the metabolic route, variation was simulated based on kinetic constants obtained from incubations with a range of individual human liver fractions or by combining kinetic constants obtained for specific isoenzymes with literature reported human variation in the activity of these enzymes. The results of the study indicate that formation of 1'-sulfooxymethyleugenol is predominantly affected by variation in i) P450 1A2-catalyzed bioactivation of methyleugenol to 1'-hydroxymethyleugenol, ii) P450 2B6-catalyzed epoxidation of methyleugenol, iii) the apparent kinetic constants for oxidation of 1'-hydroxymethyleugenol, and iv) the apparent kinetic constants for sulfation of 1'-hydroxymethyleugenol. Based on the Monte Carlo simulations a so-called chemical-specific adjustment factor (CSAF) for intraspecies variation could be derived by dividing different percentiles by the 50th percentile of the predicted population distribution for 1'-sulfooxymethyleugenol formation. The obtained CSAF value at the 90th percentile was 3.2, indicating that the default uncertainty factor of 3.16 for human variability in kinetics may adequately cover the variation within 90% of the population. Covering 99% of the population requires a larger uncertainty factor of 6.4. In conclusion, the results showed that adequate predictions on interindividual human variation can be made with Monte Carlo-based PBK modeling. For methyleugenol this variation was observed to be in line with the default variation generally assumed in risk assessment.

Determination of the exposure parameters that maximise the concentrations of the anaesthetic/sedative eugenol in rainbow trout (Oncorhynchus mykiss) skin-on fillet tissue.

Studies were conducted to determine the anaesthetic/sedative concentrations and durations that would maximise anaesthetic/sedative residue concentrations in rainbow trout (Oncorhynchus mykiss) skin-on fillet tissue. Rainbow trout (167-404 g) were exposed to 50 mg l(-1) AQUI-S(®) 20E (10% active ingredient, eugenol) in 17°C freshwater for durations up to 1440 min, 100 and 250 mg l(-1) AQUI-S(®) 20E for durations up to 240 min, and 500 and 1000 mg l(-1) AQUI-S(®) 20E for durations up to 90 min. Fish exposed to 100 mg l(-1) AQUI-S(®) 20E for durations of 30, 60, 120 and 240 min had the greatest eugenol concentrations in the fillet tissue, 50, 58, 54 and 62 µg g(-1), respectively. All other exposure concentrations and durations resulted in significantly lower eugenol concentrations, i.e. all < 39 µg g(-1).