Intranasal administration of perillyl alcohol-loaded nanoemulsion and pharmacokinetic study of its metabolite perillic acid in plasma and brain of rats using ultra-performance liquid chromatography/tandem mass spectrometry.

Perillyl alcohol (POH) is a monocyclic terpene that has strong antitumor activity. Brain tumors are particularly difficult to treat with therapeutic agents, and clinical trials have shown their low tolerance through oral administration. We proposed the entrapment of POH into an oil-in-water chitosan nanoemulsion aiming its intranasal administration for brain targeting. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitation of total metabolite perillic acid (PA) in plasma and brain of rats. The rat samples containing the metabolite were treated by liquid-liquid extraction with acetonitrile. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B), at a flow rate of 0.3 mL min-1 in gradient elution. The chromatography was run for 10 min, and analytical curves were built in acetonitrile, plasma, and brain. The PA was detected in positive ion mode with multiple reaction monitoring. The method has shown high selectivity, sensitivity, and throughput. The low quantification limits of 162, 178, and 121 ng mL-1 for acetonitrile, brain, and plasma, respectively, indicate a good detectability of the method. The repeatability and precision observed were within the limits recommended in the literature. The accuracy of the method was verified through high recovery rates (106-118%). The validated method was successfully applied to the pharmacokinetic study of the metabolite PA after the intranasal administration of free or POH-loaded nanoemulsion in rats. The results showed that chitosan nanoemulsion improved the plasma and brain bioavailability of POH, representing a promising alternative to free POH treatment.

Marine biomaterials: Biomimetic and pharmacological potential of cultivated Aplysina aerophoba marine demosponge.

Marine demosponges of the Verongiida order are considered a gold-mine for bioinspired materials science and marine pharmacology. The aim of this work was to simultaneously isolate selected bromotyrosines and unique chitinous structures from A. aerophoba and to propose these molecules and biomaterials for possible application as antibacterial and antitumor compounds and as ready-to-use scaffolds for cultivation of cardiomyocytes, respectively. Among the extracted bromotyrosines, the attention has been focused on aeroplysinin-1 that showed interesting unexpected growth inhibition properties for some Gram-negative clinical multi-resistant bacterial strains, such as A. baumannii and K. pneumoniae, and on aeroplysinin-1 and on isofistularin-3 for their anti-tumorigenic activity. For both compounds, the effects are cell line dependent, with significant growth inhibition activity on the neuroblastoma cell line SH-SY5Y by aeroplysinin-1 and on breast cancer cell line MCF-7 by isofistularin-3. In this study, we also compared the cultivation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) on the A. aerophoba chitinous scaffolds, in comparison to chitin structures that were pre-coated with Geltrex™, an extracellular matrix mimetic which is used to enhance iPSC-CM adhesion. The iPSC-CMs on uncoated and pure chitin structures started contracting 24 h after seeding, with comparable behaviour observed on Geltrex-coated cell culture plates, confirming the biocompatibility of the sponge biomaterial with this cell type. The advantage of A. aerophoba is that this source organism does not need to be collected in large quantities to supply the necessary amount for further pre-clinical studies before chemical synthesis of the active compounds will be available. A preliminary analysis of marine sponge bioeconomy as a perspective direction for application of biomaterials and secondary bioactive metabolites has been finally performed for the first time.

Simultaneous measurement of perillyl alcohol and its metabolite perillic acid in plasma and lung after inhalational administration in Wistar rats.

The inhalational administration of drugs is a practical and non-invasive approach with the potential to reduce side effects and with a quick onset of therapeutic activity. Perillyl alcohol (POH) is a monoterpene with antitumor activity that currently is undergoing clinical evaluation as an inhalational anticancer agent. A detection method was developed that will be applicable to pharmacokinetic studies of not only POH, but also its longer-lived main metabolite, perillic acid (PA), in lung tissue and plasma after inhalational delivery. The anticancer activity of POH was investigated in vitro with the use of various lung cancer cell lines. Toxicity was established by a standard MTT assay, and apoptosis markers were analyzed by Western blot. For the detection of POH and PA in lungs and plasma, albino Wistar rats were used that were exposed to POH inhalation. Tissues were subjected to chromatographic separation on an Agilent Zorbax Eclipse XDB C18 column, followed by detection of absorption in the ultraviolet (UV) range. In vitro, POH exerted cytotoxic activity against six different lung tumor cell lines, and apoptotic cell death was indicated by induction of active caspase 3 and cleavage of poly (ADP-ribose) polymerase 1 (PARP1). These results demonstrate that inhalational delivery of POH results in effective biodistribution and metabolism of POH in the systemic circulation. In addition, our study introduces a simple, rapid HPLC-UV method with high accuracy for simultaneous detection of POH and its metabolite PA in plasma, and for sensitive detection of PA in lung tissue, which should prove useful for applications in clinical studies.

Human breast tissue disposition and bioactivity of limonene in women with early-stage breast cancer.

Limonene is a bioactive food component found in citrus peel oil that has shown chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open-label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited 43 women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for two to six weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean = 41.3 µg/g tissue), whereas the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P = 0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase-3 expression. No significant changes in serum leptin, adiponectin, TGF-ß1, insulin-like growth factor binding protein-3 (IGFBP-3), and interleukin-6 (IL-6) levels were observed following limonene intervention. There was a small but statistically significant postintervention increase in insulin-like growth factor I (IGF-I) levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell-cycle arrest and reduced cell proliferation. Furthermore, placebo-controlled clinical trials and translational research are warranted to establish limonene's role for breast cancer prevention or treatment.

Organ accumulation in mice after inhalation of single or mixed essential oil compounds.

Essential oils are composed of multiple components. It is thought that the effect of essential oils is due to specific component ratios, which may differ from the original ratio when the essential oil is absorbed. However, very little detailed research exists in this area. We studied the distribution of essential oil components after inhalation of single and mixed components in mice. This research was done using four main components of Alpinia zerumbet (Pers.) B. L. Burtt. and R. M. Sm.: α-pinene, p-cymene, 1,8-cineole, and limonene. After inhalation of single or mixed components for 90 min, component levels in the brain and liver of mice were measured. The results indicated that the amount of α-pinene in the brain and liver was twofold greater after mixed-component inhalation than that after single-component inhalation. In a comparison of the components of the mixed inhalation, the ratio of α-pinene increased to about three times that of 1,8-cineole. It is thought that the absorption via the nasal mucus greatly influences this phenomenon. The results of this investigation of the bodily distribution of essential oil volatile components may provide clues for elucidating their action.

Effect of orange oil on the oral absorption of enrofloxacin in rats.

This study was conducted to evaluate the oral absorption of enrofloxacin (ENFX) in rats when administered with orange oil or its main component, limonene. Compared with the group administered ENFX alone, the ENFX + limonene group did not show any significant difference in the absorption of ENFX, whereas the extent and rate of absorption of ENFX were significantly decreased in the ENFX + orange oil group (C(max), -43%; T(max), 129%). In addition, t(1/2λz) and MRT of ENFX were prolonged by the concomitant administration of orange oil. The AUCs of ENFX were not affected in the ENFX + orange oil group. These results suggest that decreased oral absorption could reduce the efficacy of ENFX therapy in animals.

Bioequivalence evaluation of two D-limonene capsule formulations in healthy Chinese volunteers.

A randomized, three-period crossover study was conducted in 24 healthy Chinese male volunteers to compare the bioavailability of two brands of D-limonene (0.3 ml) capsules, and determine the plasma concentration of endogenous D-limonene in food-controlled non-treated humans. The three kinds of treatments were administration of the reference formulation, administration of the test, and non-administration. The plasma samples were analyzed by a validated GC-MS method after liquid-liquid extraction. The pharmacokinetic parameters AUC(0-t), AUC(0-infinity), Cmax, tmax, and t1/2 were determined from the concentration-time profiles for both formulations and were compared statistically to evaluate bioequivalence between the two brands. The analysis of variance (ANOVA) did not show any significant difference between the two formulations and 90% confidence intervals fell within an acceptable range for bioequivalence. Besides, for the food-controlled non-treated volunteers, their plasma concentrations of D-limonene were detectable and kept relatively steady (2.94 +/- 1.38 ng/ml) within the sample collection period. Based on the statistical analysis, it was concluded that the two D-limonene capsule formulations were bioequivalent.

Tetrahydro anthranilic acid as a surrogate for anthranilic acid: application to the discovery of potent niacin receptor agonists.

The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described. This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were identified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and CYP2C9, and improved oral exposure in mice.

Anti-inflammatory senescence actives 5203-L molecule to promote healthy aging and prolongation of lifespan.

The aging process depends on genetic stability, metabolic control, and resistance to stress; longevity in particular seems related to resistance to stress. Responses to stress anticipate adaptation to an unacceptable disparity between real or imagined personal experience and expectation, including adaptive stress, anxiety, and depression. However, if stress persists, it may lead to chronic diseases, ranging from inflammation and cancer to degenerative diseases. For some time, only remarkable stress was acknowledged to induce immune and vascular alterations, such as infection or hypertension. Now it is known that moderate stress independent of conventional risk factors can induce a potent alteration of health conditions and consequently shorten life quality and lifespan. Inflammation is a critical defense mechanism, that, uncontrolled, contributes to chronic conditions with inflammatory pathogenesis. Stressful life conditions turn out to induce a diffuse (systemic) pro-inflammatory status. Subclinical chronic inflammation is an important pathogenic factor in the development of metabolic syndrome, a cluster of common pathologies, including cardiovascular disease. Markers include mediators associated with endothelial activation and dysfunction. This work reports the in vitro and in vivo effects of the monoterpene AISA 5203-L on human vascular endothelial cells in reversing replicative senescence in preventing and alleviating nonpathological stress, as assessed by a functional observational battery (FOB) of 44 tests, addressing behavioral, neurological, and physiological criteria.

Gas chromatographic-mass spectrometric analysis of d-limonene in human plasma.

d-Limonene shows carminative and cholagogue effects and is used in treatment of gallstone, cholecystitis and angiocholitis. A simple method was developed to determine the concentration of d-limonene in human plasma. d-Limonene and internal standard (naphthalane, C(10)H(18)) were extracted with n-hexane and then injected to GC-MS. Calibration curves were linear (r=0.9990, n=6) in the range of 2-500 ng/ml for d-limonene in human plasma. Limit of detection and quantification were 0.5 and 2 ng/ml, respectively. This rapid and specific method was applied to the clinical pharmacokinetic investigation of d-limonene.