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Background: The antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA). Methodology/Principal Findings: The effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH) activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC50 values of 17 and 18 mu M, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC50 value (for the inhibitable component) of 34 mu M. Conclusions/Significance: The present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer

Increased Expression of Cannabinoid CB1 Receptors in Achilles Tendinosis

BACKGROUND: The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB₁) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis. METHODOLOGY: Cannabinoid CB₁ receptor immunoreactivity (CB₁IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons. PRINCIPAL FINDINGS: CB₁IR was seen as a granular pattern in the tenocytes. CB₁IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB₁ receptor expression in tendinosis tissue compared to control tissue. CONCLUSION: Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder

The endocannabinoid system : a translational study from Achilles tendinosis to cyclooxygenase

The endogenous cannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert their effect by activating cannabinoid receptors (CB). These receptors mediate a broad range of physiological functions such as beneficial effects in pain and inflammation, although little is known about the expression of CB receptors in human pain conditions. AEA and 2-AG are short- lived molecules due to their rapid cellular accumulation and metabolism. The enzymes primarily responsible for their degradation are fatty acid amide hydrolase (FAAH) for AEA and monoacylglycerol lipase (MGL) for 2-AG. Inhibition of endocannabinoid metabolism is a potential approach for drug development, and there is a need for the identification of novel compounds with inhibitory effects upon FAAH and MGL. In Paper I of this thesis, the expression of CB1 receptors in human Achilles tendon was examined. We found expression of CB1 receptors in tenocytes, blood vessel wall as well as in the perineurium of the nerve. A semi-quantitative analysis showed an increase of CB1 receptors in painful human Achilles tendinosis. In papers II and III, termination of AEA signalling was investigated via inhibition of FAAH. In Paper II, Flu-AM1, an analogue of flurbiprofen, was investigated. The compound inhibited both FAAH and the oxygenation of 2-AG by cyclooxygenase-2. In Paper III the antifungal compound ketoconazole was shown to inhibit the cellular uptake of AEA in HepG2, CaCo-2 and C6 cell lines in a manner consistent with inhibition of FAAH. The role of FAAH in gating the cellular accumulation of AEA was investigated in Paper IV. FAAH has been shown to control the concentration gradient of AEA across the plasmamembrane in RBL2H3 cells, whereas no such effect is seen in other FAAH-expressing cell lines. To determine whether this effect is assay dependent or due to intrinsic differences between the cell lines, we assayed four cell lines with different levels of FAAH expression using the same methodology. We found that the sensitivity of FAAH uptake inhibition was not dependent on the expression level of FAAH, suggesting that factors other than FAAH are important for uptake. Paper V is focused on the inhibition of MGL. Prior to this study no selective inhibitors of the enzyme had been described. Thus, we screened a number of compounds for their inhibitory effect on MGL. Troglitazone was found to be an inhibitor of MGL, although its potency was dependent upon the enzyme assay used.

Involvement of Fatty Acid Amide Hydrolase and Fatty Acid Binding Protein 5 in the Uptake of Anandamide by Cell Lines with Different Levels of Fatty Acid Amide Hydrolase Expression: A Pharmacological Study

Background: The endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process ofcellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces theobserved uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentrationgradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differencesare methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the roleof FAAH in gating the uptake of AEA.Methodology/Principal Findings: The effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. SemiquantitativePCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of atranscript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four couldaccumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in thesensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor,with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. Thefour cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggestingthat the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5.Conclusions/Significance: When assayed using the same methodology, different FAAH-expressing cells display differentsensitivities of uptake to FAAH inhibition

Immunofluorescence for CB₁ in blood vessel and nerve fasicle.

<p>Panels show sections of Achilles tendinosis tissue showing a small blood vessel (<b>a,b</b>) and a part of a nerve fascicle (<b>c,d</b>) stained with htx-eosin (<b>a</b>), processed for CB₁IR (<b>b,d</b>), and for PGP9.5 (<b>c</b>). Immunoreactions (arrows) are seen in the blood vessel wall (<b>b</b>) and in the perineurium of the nerve fascicle (arrows) (d). Original magnification ×20 (<b>a</b>), ×40 (<b>c,d</b>), ×63 (<b>b</b>). Asterisks in similar region in (<b>a</b>) and (<b>b</b>) and in the perineurium in (<b>c</b>) and (<b>d</b>).</p

Immunofluorescence for CB₁ in Achilles tendinosis.

<p>Panels show sections of Achilles tendinosis tendons processed for demonstration of CB₁ (<b>a,c,d</b>) and of CB₁ after preabsorption with the immunogenic peptide (<b>b</b>). Numerous tenocytes are seen in low magnification in (<b>a</b>) and (<b>b</b>) (arrows at tenocytes). They show specific immunoreactions in (<b>a</b>) but not in (<b>b</b>). In panel <b>c</b> and <b>d</b> in which the tenocytes are shown in high magnification, punctuate immunoreactions in tenocytes are shown (arrows). Original magnification ×20 (<b>a,b</b>), ×63 (<b>c,d</b>).</p

CB₁IR scores for biopsy samples from controls and patients with Achilles tendinopathy.

<p>Shown is a box and whiskers plot of CB₁ immunoreactivity in pain-free Achilles tendons (controls, n = 7) vs. tendons from patients with Achilles tendinopathy (n = 17). *p = <0.05, two-tailed Mann-Whitney U test. Values are mean of the scoring made by the two investigators.</p

Expression of FAAH and sensitivity of AEA uptake to the FAAH inhibitor URB597 in AT1, C6, RBL-2H3 and P19 cells.

<p>In Panels A and B semi-quantitative PCR analysis of the mRNA expression of FAAH are shown for the cells, with rat brain lysates (two different lysates, lanes 2–5, in Panel B) as positive controls. Molecular size markers are also shown. Total RNA was isolated and reverse transcribed into cDNA. Samples were pooled (n = 4) and the PCR analysis was performed using primers designed to recognize rat or mouse FAAH, as appropriate. The PCR products were analyzed by agarose gel electrophoresis and fragment size estimated using a 100 bp marker. The arrows show the expected sizes for FAAH (both Panels) and FLAT (Panel B) with the primer pair used. In Panel B, the main gel shows an overexposure of the gels, with the small panel above showing the band corresponding to FAAH at normal levels of exposure. The photographs have been inverted to show the minor bands. In Panel C, the effect of 1 µM URB597 upon the uptake of [³H]AEA is shown for AT1, C6, RBL2H3 and P19 cells. Cells (or wells alone) were preincubated with URB597 for 10 min 37°C followed by addition of 100 nM [³H]AEA and incubation for further 4 min at 37°C. Shown are means and s.e.m., n = 5. The statistical treatment of the data is presented in Results.</p

Effects of ketoconazole, nefazodone, AM404 and URB597 (“U”, 1 µM) upon A, the uptake of [³H]AEA; B, the hydrolysis of [³H]AEA and C, the hydrolysis of [³H]2-AG by C6 glioma cells.

<p>The compounds were preincubated with the cells for 10-AG (100 nM final concentration) and incubation for a further 10 min at 37°C. For the uptake experiments, the tritium label was on the arachidonoyl side chain, whilst for the hydrolysis experiments, the label was on the ethanolamine group (AEA) or the glycerol group (2-AG). Shown are means ± s.e.m. (when not enclosed by the symbols), n = 3, except for ketoconazole in Panel B, where n = 4.</p

Effect of compound 33 upon AEA uptake using either [³H-ethanolamine]- or [³H-arachidonoyl]- labelled ligand and hydrolysis of [³H-ethanolamine]-AEA by cell lysates.

<p>The same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103479#pone-0103479-g002" target="_blank">figure 2</a> were used. Shown are means ± s.e.m., n = 3–4. The data for [³H-arachidonoyl]- labelled AEA uptake is the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103479#pone-0103479-g002" target="_blank">Fig. 2</a>, but in this case the x-axes are for cell lysates rather than intact cells. The values (± s.e.m.) of the slopes determined from the regression lines of the pooled data for [³H-ethanolamine]- or [³H-arachidonoyl]- labelled AEA, respectively, were: C6 cells,, 1.03±0.10 and 1.11±0.31; RBL-2H3 cells, 0.49±0.14 and 0.46±0.14. The concentration of EtOH for the vehicle was 0.2% (cells) and 1% (lysates).</p