In Vitro Metabolism and CYP-Modulating Activity of Lavender Oil and Its Major Constituents.

The application of essential oils has historically been limited to topical (massage therapy) and inhalational (aromatherapy) routes of administration. More recently, however, evaluation of the therapeutic effects of essential oils has expanded to include the oral route of administration, which increases the herb-drug interaction potential. The purpose of this study was to evaluate the herb-drug interaction potential of lavender essential oil and two of its primary phytoactive constituents, namely linalool and linalyl acetate. The metabolic stability of linalool and linalyl acetate was determined in human liver microsomes (HLM) and S9 fractions by quantitative analysis using UPLC-MS/MS system. Linalool was metabolically unstable in HLM and S9 fractions with an intrinsic clearance of 31.28 mL·min-1·kg-1, and 7.64 mL·min-1·kg-1, respectively. Interestingly, it was observed that linalyl acetate converted to linalool both in HLM and S9 fractions. Lavender oil showed weak inhibitory effect on the catalytic activity of CYP3A4 and CYP1A2 enzymes (IC50 12.0 and 21.5 µg/mL). Linalyl acetate inhibited CYP3A4 (IC50 4.75 µg/mL) while linalool did not show any inhibitory effect on any of the enzymes. The lavender oil and its constituents did not activate PXR to a considerable extent, and no activation of AhR was observed, suggesting a lack of potential to modify the pharmacokinetic and pharmacodynamic properties of conventional medications if used concurrently.

Development of a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for Characterizing Linalool Oral Pharmacokinetics in Humans.

Lavender (Lavandula angustifolia Miller or Lavandula officinalis Chaix) is an ethnopharmacological plant commonly known as English lavender. Linalool and linalyl acetate are putative phytoactives in lavender essential oil (LEO) derived from the flower heads. LEO has been used in aroma or massage therapy to reduce sleep disturbance and to mitigate anxiety. Recently, an oral LEO formulation was administered in human clinical trials designed to ascertain its anxiolytic effect. However, human pharmacokinetics and an LC-MS/MS method for the measurement of linalool are lacking. To address this deficiency, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of linalool in human serum. Prior to the analysis, a simple sample preparation protocol including protein precipitation and liquid-liquid extraction of serum samples was created. The prepared samples were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to quantitatively determine linalool and IS according to transitions of m/z 137.1→95.1 (tR 0.79 min) and 205.2→149.1 (tR 1.56 min), respectively. The method was validated for precision, accuracy, selectivity, linearity, sensitivity, matrix effects, and stability, and it was successfully applied to characterize the oral pharmacokinetics of linalool in humans. The newly developed LC-MS/MS-based method and its application in clinical trial serum samples are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

Development of a GC/Q-ToF-MS Method Coupled with Headspace Solid-Phase Microextraction to Evaluate the In Vitro Metabolism of ß-Caryophyllene.

Sample preparation remains both a challenging and time-consuming process in the field of bioanalytical chemistry. Many traditional techniques often require multi-step processes, which can introduce additional errors to the analytical method. Given the complexity of many biological matrices, thorough analyte extraction presents a major challenge to researchers. In the present study, a headspace solid-phase microextraction (HS-SPME) coupled with a GC/Q-ToF-MS method, was developed to quantify in vitro metabolism of ß-caryophyllene by both human liver microsome (HLM) and S9 liver fractions. Validation of the method was demonstrated both in terms of linearity (R2 = 0.9948) and sensitivity with a limit of detection of 3 ng/mL and a limit of quantitation of 10 ng/mL. In addition, the method also demonstrated both inter- and intra-day precision with the relative standard deviation (RSD) being less than 10% with four concentrations ranging from 50-500 ng/mL. Since this method requires no solvents and minimal sample preparation, it provides a rapid and economical alternative to traditional extraction techniques. The method also eliminates the need to remove salts or buffers, which are commonly present in biological matrices. Although this method was developed to quantify in vitro metabolism of one analyte, it could easily be adapted to detect or quantify numerous volatiles and/or semi-volatiles found in biological matrices.

Metabolomic Analysis Identified Reduced Levels of Xenobiotics, Oxidative Stress, and Improved Vitamin Metabolism in Smokers Switched to Vuse Electronic Nicotine Delivery System.

INTRODUCTION: Switching to noncombustible tobacco products presents an opportunity for cigarette smokers to potentially reduce the health risks associated with smoking. Electronic Nicotine Delivery Systems (ENDS) are one such product because the vapor produced from ENDS contains far fewer toxicants than cigarette smoke. To investigate the biochemical effects of switching from smoking to an ENDS, we assessed global metabolomic profiles of smokers in a 7-day confinement clinical study. METHODS: In the first 2 days of this clinical study, the subjects used their usual brand of cigarettes and then switched to exclusive ENDS ad libitum use for 5 days. Urine and plasma samples were collected at baseline and 5 days after switching. The samples were analyzed using a mass spectrometry-based metabolomic platform. RESULTS: Random forest analyses of urine and plasma metabolomic data revealed excellent predictive accuracy (>97%) of a 30-metabolite signature that can differentiate smokers from 5-day ENDS switchers. In these signatures, most biomarkers are nicotine-derived metabolites or xenobiotics. They were significantly reduced in urine and plasma, suggesting a decreased xenobiotic load on subjects. Our results also show significantly decreased levels of plasma glutathione metabolites after switching, which suggests reduced levels of oxidative stress. In addition, increased urinary and plasma levels of vitamins and antioxidants were identified, suggesting enhanced bioavailability due to discontinuation of cigarette smoking and switching to Vuse ENDS use. CONCLUSIONS: Our results suggest reduced toxicant exposure, reduced oxidative stress, and potential beneficial changes in vitamin metabolism within 5 days in smokers switching to Vuse ENDS. IMPLICATIONS: Switching from smoking to exclusive ENDS use in clinical confinement settings results in significant reduction of nicotine metabolites and other cigarette-related xenobiotics in urine and plasma of subjects. Significantly decreased oxidative stress-related metabolites and increased urinary and plasma levels of vitamin metabolites and antioxidants in 5-day short-term ENDS switchers suggest less toxic physiological environment for consumers of ENDS products and potential health benefits if such changes persist.

Genome-Wide Association Study in African Americans with Acute Respiratory Distress Syndrome Identifies the Selectin P Ligand Gene as a Risk Factor.

RATIONALE: Genetic factors are involved in acute respiratory distress syndrome (ARDS) susceptibility. Identification of novel candidate genes associated with increased risk and severity will improve our understanding of ARDS pathophysiology and enhance efforts to develop novel preventive and therapeutic approaches. OBJECTIVES: To identify genetic susceptibility targets for ARDS. METHODS: A genome-wide association study was performed on 232 African American patients with ARDS and 162 at-risk control subjects. The Identify Candidate Causal SNPs and Pathways platform was used to infer the association of known gene sets with the top prioritized intragenic SNPs. Preclinical validation of SELPLG (selectin P ligand gene) was performed using mouse models of LPS- and ventilator-induced lung injury. Exonic variation within SELPLG distinguishing patients with ARDS from sepsis control subjects was confirmed in an independent cohort. MEASUREMENTS AND MAIN RESULTS: Pathway prioritization analysis identified a nonsynonymous coding SNP (rs2228315) within SELPLG, encoding P-selectin glycoprotein ligand 1, to be associated with increased susceptibility. In an independent cohort, two exonic SELPLG SNPs were significantly associated with ARDS susceptibility. Additional support for SELPLG as an ARDS candidate gene was derived from preclinical ARDS models where SELPLG gene expression in lung tissues was significantly increased in both ventilator-induced (twofold increase) and LPS-induced (5.7-fold increase) murine lung injury models compared with controls. Furthermore, Selplg-/- mice exhibited significantly reduced LPS-induced inflammatory lung injury compared with wild-type C57/B6 mice. Finally, an antibody that neutralizes P-selectin glycoprotein ligand 1 significantly attenuated LPS-induced lung inflammation. CONCLUSIONS: These findings identify SELPLG as a novel ARDS susceptibility gene among individuals of European and African descent.

Novel Small Molecule JP-153 Targets the Src-FAK-Paxillin Signaling Complex to Inhibit VEGF-Induced Retinal Angiogenesis.

Targeting vascular endothelial growth factor (VEGF) is a common treatment strategy for neovascular eye disease, a major cause of vision loss in diabetic retinopathy and age-related macular degeneration. However, the decline in clinical efficacy over time in many patients suggests that monotherapy of anti-VEGF protein therapeutics may benefit from adjunctive treatments. Our previous work has shown that through decreased activation of the cytoskeletal protein paxillin, growth factor-induced ischemic retinopathy in the murine oxygen-induced retinopathy model could be inhibited. In this study, we demonstrated that VEGF-dependent activation of the Src/FAK/paxillin signalsome is required for human retinal endothelial cell migration and proliferation. Specifically, the disruption of focal adhesion kinase (FAK) and paxillin interactions using the small molecule JP-153 inhibited Src-dependent phosphorylation of paxillin (Y118) and downstream activation of Akt (S473), resulting in reduced migration and proliferation of retinal endothelial cells stimulated with VEGF. However, this effect did not prevent the initial activation of either Src or FAK. Furthermore, topical application of a JP-153-loaded microemulsion affected the hallmark features of pathologic retinal angiogenesis, reducing neovascular tuft formation and increased avascular area, in a dose-dependent manner. In conclusion, our results suggest that using small molecules to modulate the focal adhesion protein paxillin is an effective strategy for treating pathologic retinal neovascularization. To our knowledge, this is the first paradigm validating modulation of paxillin to inhibit angiogenesis. As such, we have identified and developed a novel class of small molecules aimed at targeting focal adhesion protein interactions that are essential for pathologic neovascularization in the eye.

Mitigation of radiation injury by selective stimulation of the LPA(2) receptor.

Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA(2) receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA(2) receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24h after radiation exposure. Our findings suggest that by specifically activating LPA(2) receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Quinic acid derivative KZ-41 exhibits radiomitigating activity in preclinical models of radiation injury.

Acute radiation syndrome is induced when a significant portion of the body receives high-dose, as well as high-dose rate, radiation. We have previously identified a quinic acid-based derivative, KZ-41, that protects from radiation injury. Further preclinical efficacy studies were conducted to determine the radiomitigating activity of KZ-41. C57BL/6 mice received total body irradiation (TBI-LD80/30, ¹³7Cs; ∼2 min) followed by either normal saline or KZ-41 (100 mg/kg sc ∼26 h post-TBI). KZ-41 increased 30-day survival by approximately 45% compared with vehicle controls (P < 0.05). To further investigate the potential radiomodulating mechanisms of KZ-41, we developed a combined radiation and vascular injury model. C57BL/6 mice surgically fixed with dorsal windows for dermal vasculature imaging received either sham or TBI (¹³7Cs; 6 Gray). Postcapillary venule injury was induced (24, 48, 72, and 96 h post-TBI) followed by imaging at 5 min and 24 h to assess clot formation and blood flow. Impairment in flow (P < 0.05) and clot formation (P < 0.05) were observed as early as 48 and 72 h, respectively. Thus, vascular injury 72 h post-TBI was used to evaluate intervention (KZ-41; 100 mg/kg i.p. at 12, 36, and 60 h post-TBI) on radiation-induced changes in both flow and clot formation. KZ-41, although not improving flow, increased clot formation (P < 0.05). Platelet counts were lower in both irradiated groups compared with sham controls (P < 0.05). In summary, KZ-41 exerts radiomitigating activity in lethally irradiated mice. Imaging results suggest KZ-41 exerts radiomitigating activity through mechanisms involving promotion of initial clot formation and vascular flow restoration. The imaging model described herein is useful for further examination of radiation-induced vascular injury repair mechanisms.

LC-MS/MS identification of the one-carbon cycle metabolites in human plasma.

The one-carbon cycle is composed of four major biologically important molecules: methionine (L-Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and homocysteine (Hcy). In addition to these key metabolites, there are multiple enzymes, vitamins, and cofactors that play essential roles in the cascade of the biochemical reactions that convert one metabolite into another in the cycle. Simultaneous quantitative measurement of four major metabolites can be used to detect possible aberrations in this vital cycle. Abnormalities in the one-carbon cycle might lead to hyper- or hypomethylation, homocystinemia, liver dysfunction, and accumulation of white-matter hyperintensities in the human brain. Previously published methods describe evaluation of several components of the one-carbon cycle, but none to our knowledge demonstrated simultaneous measurement of all four key molecules (L-Met, SAM, SAH, and Hcy). We describe a novel analytical method suitable for simultaneous identification and quantification of L-Met, SAM, SAH, and Hcy with LC-MS/MS. Moreover, we tested this method to identify these metabolites in human plasma collected from patients with multiple sclerosis and healthy individuals. In a pilot feasibility study, our results indicate that patients with multiple sclerosis showed abnormalities in the one-carbon cycle.

In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1.

We define previously unrecognized in vivo pathways of vitamin D(3) (D3) metabolism generating novel D3-hydroxyderivatives different from 25-hydroxyvitamin D(3) [25(OH)D3] and 1,25(OH)(2)D3. Their novel products include 20-hydroxyvitamin D(3) [20(OH)D3], 22(OH)D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, 1,20(OH)(2)D3, 1,20,23(OH)(3)D3, and 17,20,23(OH)(3)D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ∼20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α-hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)(2)D3 → 17,20,23(OH)(3)D3 with further 1α-hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)(2)D3 and D3 → 22(OH)D3 → 20,22(OH)(2)D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)(2)D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, and 17,20,23(OH)(3)D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)(2)D3.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing pomegranate extract pharmacokinetics in humans.

Pomegranate extracts standardized to punicalagins are a rich source of ellagitannins including ellagic acid (EA). Recent evidence suggests that gut microbiota-derived urolithin (Uro) metabolites of ellagitannins are pharmacologically active. Studies have evaluated the pharmacokinetics of EA, however, little is known about the disposition of urolithin metabolites (urolithin A (UA) and B (UB)). To address this gap, we developed and applied a novel ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the characterization of EA and Uro oral pharmacokinetics in humans. Subjects (10/cohort) received a single oral dose (250 or 1000 mg) of pomegranate extract (Pomella® extract) standardized to contain not less than 30 % punicalagins, < 5 % EA, and not less than 50 % polyphenols. Plasma samples, collected over 48 h, were treated with ß-glucuronidase and sulfatase to permit comparison between unconjugated and conjugated forms of EA, UA and UB. EA and urolithins were separated by gradient elution (acetonitrile/water, 0.1 % formic acid) using a C18 column connected to a triple quadrupole mass spectrometer operating in the negative mode. Conjugated EA exposure was ∼5-8-fold higher than unconjugated EA for both dose groups. Conjugated UA was readily detectable beginning ∼8 h post-dosing, however, unconjugated UA was detectable in only a few subjects. Neither form of UB was detected. Together these data indicate EA is rapidly absorbed and conjugated following oral administration of Pomella® extract. Moreover, UA's delayed appearance in the blood, primarily in the conjugated form, is consistent with gut microbiota-mediated metabolism of EA to UA, which is then rapidly converted to its conjugated form.

Tinospora Cordifolia: A review of its immunomodulatory properties.

Emergent health threats have heightened human awareness of the need for health and wellness measures that promote resilience to disease. In addition to proper nutrition and exercise, health-conscious consumers are seeking natural-based modalities, e.g. botanical preparations, that positively impact the immune system. In Ayurvedic ethnomedicine, Tinospora cordifolia (T. cordifolia), a deciduous climbing shrub indigenous to India, has been used to historically to combat acute and chronic inflammation as well as to promote a balanced immune response. As a dietary supplement, T. cordifolia has been administered most often as a decoction either alone or in compositions containing other medicinal plant extracts of the Terminalia and Phyllanthus species. Extensive phytochemical characterization of aqueous and alcoholic extracts of different Tinospora species has identified over two hundred different phytochemicals from non-overlapping chemical classes with the most abundant being diterpenoids containing the clerodane-type skeleton. Numerous pharmacology studies have demonstrated that T. cordifolia modulates key signaling pathways related to cell proliferation, inflammation, and immunomodulation. However, rigorous dereplication studies to identify active constituents in various T. cordifolia extracts and their fractions are lacking. In this review, we will summarize the current information regarding T. cordifolia's ethnomedicinal uses, phytochemistry, pharmacological activities, and safety in order to highlight its potential as an immunomodulatory dietary supplement.

Methylsulfonylmethane and Sesame Seed Oil Improve Dyslipidemia and Modulate Polyunsaturated Fatty Acid Metabolism in Two Mouse Models of Diabetes.

The objective of this study was to identify alterations in lipids and polyunsaturated fatty acid (PUFA) metabolism in both the streptozotocin (STZ)-induced type 1 diabetic (T1D) mouse and the mutant db/db type 2 diabetic (T2D) mouse to establish a biological signature for the evaluation of natural products with purported lipid-altering activity. Eight-week-old male C57BL/6J mice were randomized to nondiabetic group or STZ-induced diabetic groups (n = 10/group). STZ-induced diabetic mice and 6-week-old male db/db mice (n = 10/group) were randomized to the following groups: (1) diabetic control, no treatment, (2) methylsulfonylmethane (MSM) treatment, (3) sesame seed oil (SSO) treatment, and (4) MSM+SSO combination treatment. Clinical parameters measured included weights, blood glucose, serum lipid panels, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of free fatty acids in serum, liver, brain, and eyes. Blood glucose significantly decreased after 4 weeks of MSM treatment in T1D mice. Serum PUFA levels were significantly reduced in T2D mice compared with control mice. In contrast, treatment with SSO reversed this effect in T2D mice, exhibiting serum PUFA levels comparable to control mice. Serum triglycerides were significantly increased in both diabetic models compared to nondiabetic control, mimicking diabetes in people. High-density lipoprotein (HDL) was significantly increased in T1D receiving MSM+SSO and all T2D treatment groups. A corresponding significant decrease in non-HDL cholesterol was seen in T2D mice in all treatment groups. MSM+SSO treatment's effects on HDL and non-HDL cholesterol and PUFA metabolism could lead to improved clinical outcomes in diabetics by improving the lipid profile.

Preclinical pharmacokinetics of the radiomitigator KZ-41 in rats.

KZ-41, a quinic acid derivative, significantly reduces mortality in a murine model of hematopoietic acute radiation syndrome. The purpose of this study was to evaluate the systemic pharmacokinetics, elimination, and oral bioavailability of KZ-41 in rats. Male Sprague-Dawley rats (n = 6 per group) received a single dose (10 mg/kg) of KZ-41 administered either intravenously via the jugular vein or orally via gavage. In vitro stability was determined using both rat liver microsomes and the bacteria Gluconobacter oxydans. KZ-41 concentrations were determined using LC-MS/MS (liquid chromatography tandom mass spectrometry). Half-life of KZ-41 was ≈3 hr after either intravenous or oral administration. Mean volume of distribution was 3.3 L/kg. Extent of absorption (F) after oral administration was estimated to be ~100%, which was consistent with the finding that KZ-41 was stable to liver microsomal and bacterial degradation. Following intravenous administration, KZ-41 demonstrated a medium clearance and volume of distribution with a terminal half-life of ≈3 hr. KZ-41 was rapidly and completely absorbed (F ≅ 1), which was consistent with the findings that KZ-41 is resistant to presystemic elimination mechanisms (i.e. enteric bacterial degradation and hepatic metabolism). Thus, KZ-41 represents an excellent candidate for further development as an orally available agent for the mitigation of radiation injury.

Pharmacokinetics of a combination of Δ9-tetrahydro-cannabinol and celecoxib in a porcine model of hemorrhagic shock.

Hemorrhagic shock involves loss of a substantial portion of circulating blood volume leading to diminished cardiac output and oxygen delivery to peripheral tissues. In situations where an immediate resuscitation cannot be provided, pharmacotherapy with a novel combination of Δ9-tetrahydro-cannabinol (THC) and celecoxib (CEL) is currently investigated as an alternative strategy to prevent organ damage. In the present study, 28 Yorkshire×Landrace pigs were used to study the pharmacokinetics of THC and CEL in an established porcine model of hemorrhagic shock. Pigs in hemorrhagic shock received 0.5, 1 or 4 mg/kg THC and 2 mg/kg CEL, while normotensive pigs received 1 mg/kg THC and 2 mg/kg CEL by intravenous injection. THC and CEL plasma concentrations were simultaneously determined by LC-MS/MS. Pharmacokinetic parameters and their between animal variability were obtained using standard non-compartmental analysis as well as a compartmental analysis using nonlinear mixed effects modeling. The concentration-time profiles of THC and CEL followed a multi-exponential decline and their pharmacokinetics were similar in hemorrhagic shock and normotensive conditions, despite the substantial change in hemodynamics in the animals with shock. This interesting finding might be due to the pharmacologic effect of the THC/CEL combination, which is intended to maintain adequate perfusion of vital organs in shock. Overall, this study established THC and CEL pharmacokinetics in a porcine shock model and provides the basis for dose selection in further studies of THC and CEL in this indication.

Sequential Dynamics of Stearoyl-CoA Desaturase-1(SCD1)/Ligand Binding and Unbinding Mechanism: A Computational Study.

Stearoyl-CoA desaturase-1 (SCD1 or delta-9 desaturase, D9D) is a key metabolic protein that modulates cellular inflammation and stress, but overactivity of SCD1 is associated with diseases, including cancer and metabolic syndrome. This transmembrane endoplasmic reticulum protein converts saturated fatty acids into monounsaturated fatty acids, primarily stearoyl-CoA into oleoyl-CoA, which are critical products for energy metabolism and membrane composition. The present computational molecular dynamics study characterizes the molecular dynamics of SCD1 with substrate, product, and as an apoprotein. The modeling of SCD1:fatty acid interactions suggests that: (1) SCD1:CoA moiety interactions open the substrate-binding tunnel, (2) SCD1 stabilizes a substrate conformation favorable for desaturation, and (3) SCD1:product interactions result in an opening of the tunnel, possibly allowing product exit into the surrounding membrane. Together, these results describe a highly dynamic series of SCD1 conformations resulting from the enzyme:cofactor:substrate interplay that inform drug-discovery efforts.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing caffeine, methylliberine, and theacrine pharmacokinetics in humans.

Coffea liberica possesses stimulant properties without accumulating the methylxanthine caffeine. The basis for this peculiar observation is that methylurates (e.g., theacrine and methylliberine) have replaced caffeine. The stimulant properties of methylurates, alone and in combination with caffeine, have recently been investigated. However, human pharmacokinetics and LC-MS/MS methods for simultaneous measurement of methylxanthines and methylurates are lacking. To address this deficiency, we conducted a pharmacokinetic study in which subjects (n = 12) were orally administered caffeine (150 mg), methylliberine (Dynamine™, 100 mg), and theacrine (TeaCrine®, 50 mg) followed by blood sampling over 24 h. Liquid-liquid extraction of plasma samples containing purine alkaloids and internal standard (13C-Caffeine) were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to detect caffeine, methylliberine, theacrine, and IS transitions of m/z 195.11 â†’ 138.01, 225.12 â†’ 168.02, 225.12 â†’ 167.95, and 198.1 â†’ 140.07, respectively. The method was validated for precision, accuracy, selectivity, and linearity and was successfully applied to characterize the oral pharmacokinetics of caffeine, methylliberine, and theacrine in human plasma. Successful development and application of LC-MS/MS-based methods such as ours for the simultaneous measurement of methylxanthines and methylurates are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

Synthesis and biological evaluation of quinic acid derivatives as anti-inflammatory agents.

Quinic acid (QA) esters found in hot water extracts of Uncaria tomentosa (a.k.a. cat's claw) exert anti-inflammatory activity through mechanisms involving inhibition of the pro-inflammatory transcription factor nuclear factor kappa B (NF-kappaB). Herein, we describe the synthesis and biological testing of novel QA derivatives. Inhibition of NF-kappaB was assessed using A549 (Type II alveolar epithelial-like) cells that stably express a secreted alkaline phosphatase (SEAP) reporter driven by an NF-kappaB response element. A549-NF-kappaB cells were stimulated with TNF-alpha (10 ng/mL) in the presence or absence of QA derivative for 18 hours followed by measurement of SEAP activity. Amide substitution at the carboxylic acid position yielded potent inhibitors of NF-kappaB. A variety of modifications to the amide substitution were tolerated with the N-propyl amide derivative being the most potent. Further examination of the SAR demonstrated that acetylation of the hydroxyl groups reduced NF-kappaB inhibitory activity. QA amide derivatives lacked anti-oxidant activity and were found to be neither anti-proliferative nor cytotoxic at concentrations up to 100 microM. In conclusion, we have discovered a novel series of non-toxic QA amides that potently inhibit NF-kappaB, despite their lack of anti-oxidant activity. Mechanistic studies and pre-clinical efficacy studies in various inflammatory animal models are on-going.

Urinary Leukotriene E4 and 2,3-Dinor Thromboxane B2 Are Biomarkers of Potential Harm in Short-Term Tobacco Switching Studies.

BACKGROUND: Modified risk tobacco products (MRTP) can reduce harm by decreasing exposure to combustion-related toxicants. In the absence of epidemiologic data, biomarkers of potential harm (BoPH) are useful to evaluate the harm-reducing potential of MRTPs. This study evaluated whether arachidonic acid (AA)-derived metabolites serve as short-term BoPH for predicting harm reduction in tobacco product-switching studies. METHODS: We used 24-hour urine samples from participants in a series of short-term studies in which smokers switched from combustible to noncombustible tobacco products [oral smokeless tobacco products or electronic nicotine delivery system (ENDS)] or abstinence. Pre- and postswitching samples were analyzed by LC/MS-MS for alterations in select AA metabolites, including prostaglandins, isoprostanes, thromboxanes, and leukotrienes. RESULTS: Switching to abstinence, dual use of combustible and noncombustible products, or exclusive use of noncombustible products resulted in reduced 2,3-d-TXB2 levels. Moreover, switching smokers to either abstinence or exclusive use of oral tobacco products resulted in reduced LTE4, but dual use of combustible and oral tobacco products or ENDS did not. A two-biomarker classification model comprising 2,3-d-TXB2 and LTE4 demonstrated the highest performance in distinguishing smokers switched to either abstinence or to ENDS and oral smokeless tobacco products. CONCLUSIONS: Urinary 2,3-d-TXB2 and LTE4 can discriminate between combustible tobacco users and combustible tobacco users switched to either abstinence or noncombustible products for 5 days. IMPACT: 2,3-d-TXB2 and LTE4, which are linked to platelet activation and inflammation, represent BoPH in short-term tobacco product-switching studies. Thus, from a regulatory perspective, 2,3-d-TXB2 and LTE4 may aid in assessing the harm reduction potential of MRTPs.

Plasma and cerebrospinal fluid pharmacokinetics of the novel tetrahydroisoquinoline EDL-155 in rats.

PURPOSE: Tetrahydroisoquinolines (THIs) have demonstrated anti-cancer activity in rodent models of glioma, a form of brain cancer refractory to therapeutic intervention. In this study, peripheral and cerebrospinal fluid (CSF) pharmacokinetics in rats were determined to assess the drug developability of the novel THI EDL-155 for the treatment of glioma. METHODS: Serial blood and CSF samples were collected from rats following intravenous bolus administration of EDL-155 (10-20 mg/kg). Samples were analyzed by LC/MS/MS. Pharmacokinetic analyses using compartmental and noncompartmental methods were performed using the computer program WinNonlin. Plasma protein binding was measured using the charcoal adsorption method. The in vivo efficacy of EDL-155 (i.p. 20 mg/kg twice daily for 7 days) was assessed in rats with stereotactically implanted C6 glioma cells into the caudate. RESULTS: EDL-155 plasma concentration data were described by a one-compartment model. EDL-155 demonstrated rapid clearance (342.5+/-49.9 ml/min/kg), high volume of distribution (13.0+/-1.2 l/kg) and a terminal half-life of 23.7+/-1.5 min. Dose-normalized CSF area under the curve (AUC(CSF)) as a percentage of peripheral exposure (AUC(Plasma)) was 1.4%. EDL-155 was highly bound to plasma proteins (>93%). Intracranial tumor volume at 7 days post-implantation was approximately 30% smaller in animals treated with EDL-155 when compared to vehicle control animals (13.2+/-5.3 mm(3) vs. 18.7+/-6.3 mm(3); P=0.04). CONCLUSION: High clearance and extensive protein binding limit the brain availability of EDL-155 following systemic administration. EDL-155 treatment resulted in reduced tumor size despite limited blood brain barrier penetrability, which suggests that analogs with increased metabolic stability and brain penetrability may provide a therapeutic option for primary central nervous system tumors such as glioma. On-going studies are focused on the design, synthesis, and testing of novel analogs based upon these findings.