Abortion after deliberate Arthrotec® addition to food. Mass spectrometric detection of diclofenac, misoprostol acid, and their urinary metabolites.

Arthrotec(®) (AT) is a combination of diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), and misoprostol (MP), a synthetic analogue of prostaglandin E1 (PGE1). MP is a lipophilic methyl ester prodrug. It is readily metabolized to the biologically active misoprostol acid (MPA). During the last few years, medical studies exhibited MP to be an excellent abortive. In this paper, we describe a rare criminal case of MP abortion, initiated by the expectant father. After the abortion, samples of vomit and urine were collected. Systemic exposure to MP is difficult to prove, because both MP and the active metabolite MPA are hardly excreted in urine. Therefore, in addition to routine toxicological analysis, we used slightly modified, well-established liquid and gas chromatographic/tandem mass spectrometric (LC/MS/MS and GC/MS/MS) methods, for the direct and the indirect detection of MPA and its metabolites. In this case, we were able to demonstrate the presence of the major MP metabolites 2,3-dinor-MPA and 2,3,4,5-tetranor-MPA in the urine of the victim. We also detected paracetamol, 3-methoxyparacetamol and diclofenac-glucuronide in the urine. In the vomit of the victim, we detected diclofenac and MPA. These results, combined with the criminal investigations, showed that the accused had mixed MP into the food of his pregnant girlfriend. Finally, these investigations contributed to a confession of the accused.

A Novel Approach for Glioblastoma Treatment by Combining Apoptosis Inducers (TMZ, MTX, and Cytarabine) with E.V.A. (Eltanexor, Venetoclax, and A1210477) Inhibiting XPO1, Bcl-2, and Mcl-1.

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

Novel bioactive metabolites of dipyrone (metamizol).

Dipyrone is a common antipyretic drug and the most popular non-opioid analgesic in many countries. In spite of its long and widespread use, molecular details of its fate in the body are not fully known. We administered dipyrone orally to mice. Two unknown metabolites were found, viz. the arachidonoyl amides of the known major dipyrone metabolites, 4-methylaminoantipyrine (2) and 4-aminoantipyrine (3). They were identified by ESI-LC-MS/MS after extraction from the CNS, and comparison with reference substances prepared synthetically. The arachidonoyl amides were positively tested for cannabis receptor binding (CB(1) and CB(2)) and cyclooxygenase inhibition (COX-1 and COX-2 in tissues and as isolated enzymes), suggesting that the endogenous cannabinoid system may play a role in the effects of dipyrone against pain.

Effect of n-3 fatty acid supplementation on urinary risk factors for calcium oxalate stone formation.

PURPOSE: Findings are inconsistent in a few studies of the effect of n-3 fatty acid supplementation on urinary calcium and oxalate excretion in stone formers. We evaluated the physiological effects of supplementation with eicosapentaenoic acid and docosahexaenoic acid on urinary risk factors for calcium oxalate stone formation under standardized conditions. MATERIALS AND METHODS: We studied 15 healthy subjects initially while consuming a standardized diet for 5 days (control phase). During consecutive intervention phases 1-5-day standardized diet, 2-20-day free diet and 3-5-day standardized diet participants received 900 mg eicosapentaenoic acid and 600 mg docosahexaenoic acid daily. While ingesting the standardized diets, daily 24-hour urine samples were collected. RESULTS: After short-term supplementation with eicosapentaenoic acid and docosahexaenoic acid in phase 1 we noted no changes in urinary parameters compared to the control phase. After 30-day supplementation with eicosapentaenoic acid and docosahexaenoic acid in phase 3 relative supersaturation with calcium oxalate decreased significantly by 23% from a mean ± SD of 2.01 ± 1.26 to 1.55 ± 0.84 due to significantly decreased urinary oxalate excretion (p = 0.023). Other urinary variables were not affected by supplementation. CONCLUSIONS: Results show that 30-day n-3 fatty acid supplementation effectively decreases urinary oxalate excretion and the risk of calcium oxalate crystallization. The mechanism of the physiological effect may be decreased cellular oxalic acid exchange attributable to an altered fatty acid pattern of membrane phospholipids with concomitant changes in oxalate transporter activity. Calcium oxalate stone formers may benefit from long-term n-3 fatty acid supplementation.

A randomized comparison of pharmacokinetics of a single vaginal dose of dry misoprostol or misoprostol moistened with normal saline or with acetic acid.

BACKGROUND: The pharmacokinetics of vaginal misoprostol as a dry tablet or as a tablet moistened with normal saline or with acetic acid were studied. METHODS: For this study, 42 women requesting termination of pregnancy at gestational age of <12 weeks were recruited and received 400 µg vaginal misoprostol tablets. They were randomized into three groups: (i) dry tablets, (ii) tablets moistened with 3 ml of normal saline and (iii) tablets moistened with 3 ml of 5% acetic acid. Venous blood samples were taken at 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min after misoprostol administration. Misoprostol acid (MPA) was determined in serum samples using gas chromatography/tandem mass spectrometry. RESULTS: The serum peak MPA concentration (C(max)) was significantly higher and the time-to-peak concentration (T(max)) was significantly shorter in the normal saline and acetic acid groups, when compared with the dry tablet group. Both areas under the curve at 240 and 360 min (AUC(240) and AUC(360)) of the normal saline and acetic acid groups were also significantly greater than that of the dry tablet group. The coefficients of variation in C(max) and T(max) were highest in the normal saline group, while that of AUC(240) and AUC(360) were highest in the dry tablet group. The C(max) was significantly higher in subjects in the dry tablet group with vaginal pH < 5 than in those with pH 5. There were no significant differences in other pharmacokinetic parameters between subjects with vaginal pH < 5 and those with vaginal pH 5 in all three groups. CONCLUSIONS: Vaginal misoprostol tablets moistened with normal saline or 5% acetic acid achieved better absorption than the dry tablet. The use of vaginal misoprostol tablets moistened with normal saline or 5% acetic acid would potentially improve the clinical efficacy of misoprostol. HKClinicalTrials.com registration: HKCTR-821.

Acyl chain-dependent effect of lysophosphatidylcholine on endothelial prostacyclin production.

Previously we identified palmitoyl-lysophosphatidylcholine (16:0 LPC), linoleoyl-LPC (18:2 LPC), arachidonoyl-LPC (20:4 LPC), and oleoyl-LPC (18:1 LPC) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein. In the present study, the impact of those LPC on prostacyclin (PGI(2)) production was examined in vitro in primary human aortic endothelial cells (HAEC) and in vivo in mice. Although 18:2 LPC was inactive, 16:0, 18:1, and 20:4 LPC induced PGI(2) production in HAEC by 1.4-, 3-, and 8.3-fold, respectively. LPC-elicited 6-keto PGF1α formation depended on both cyclooxygenase (COX)-1 and COX-2 and on the activity of cytosolic phospholipase type IVA (cPLA2). The LPC-induced, cPLA2-dependent (14)C-arachidonic acid (AA) release was increased 4.5-fold with 16:0, 2-fold with 18:1, and 2.7-fold with 20:4 LPC, respectively, and related to the ability of LPC to increase cytosolic Ca(2+) concentration. In vivo, LPC increased 6-keto PGF(1α) concentration in mouse plasma with a similar order of potency as found in HAEC. Our results indicate that the tested LPC species are capable of eliciting production of PGI(2), whereby the efficacy and the relative contribution of underlying mechanisms are strongly related to acyl-chain length and degree of saturation.

15-hydroxyeicosatetraenoic acid is a preferential peroxisome proliferator-activated receptor beta/delta agonist.

Peroxisome proliferator-activated receptor (PPARs) modulate target gene expression in response to unsaturated fatty acid ligands, such as arachidonic acid (AA). Here, we report that the AA metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) activates the ligand-dependent activation domain (AF2) of PPARbeta/delta in vivo, competes with synthetic agonists in a PPARbeta/delta ligand binding assay in vitro, and triggers the interaction of PPARbeta/delta with coactivator peptides. These agonistic effects were also seen with PPARalpha and PPARgamma, but to a significantly weaker extent. We further show that 15-HETE strongly induces the expression of the bona fide PPAR target gene Angptl4 in a PPARbeta/delta-dependent manner and, conversely, that inhibition of 15-HETE synthesis reduces PPARbeta/delta transcriptional activity. Consistent with its function as an agonistic ligand, 15-HETE triggers profound changes in chromatin-associated PPARbeta/delta complexes in vivo, including the recruitment of the coactivator cAMP response element-binding protein binding protein. Both 15R-HETE and 15S-HETE are similarly potent at inducing PPARbeta/delta coactivator binding and transcriptional activation, indicating that 15-HETE enantiomers generated by different pathways function as PPARbeta/delta agonists.

α-Linolenic Acid-Rich Diet Influences Microbiota Composition and Villus Morphology of the Mouse Small Intestine.

α-Linolenic acid (ALA) is well-known for its anti-inflammatory activity. In contrast, the influence of an ALA-rich diet on intestinal microbiota composition and its impact on small intestine morphology are not fully understood. In the current study, we kept adult C57BL/6J mice for 4 weeks on an ALA-rich or control diet. Characterization of the microbial composition of the small intestine revealed that the ALA diet was associated with an enrichment in Prevotella and Parabacteroides. In contrast, taxa belonging to the Firmicutes phylum, including Lactobacillus, Clostridium cluster XIVa, Lachnospiraceae and Streptococcus, had significantly lower abundance compared to control diet. Metagenome prediction indicated an enrichment in functional pathways such as bacterial secretion system in the ALA group, whereas the two-component system and ALA metabolism pathways were downregulated. We also observed increased levels of ALA and its metabolites eicosapentanoic and docosahexanoic acid, but reduced levels of arachidonic acid in the intestinal tissue of ALA-fed mice. Furthermore, intestinal morphology in the ALA group was characterized by elongated villus structures with increased counts of epithelial cells and reduced epithelial proliferation rate. Interestingly, the ALA diet reduced relative goblet and Paneth cell counts. Of note, high-fat Western-type diet feeding resulted in a comparable adaptation of the small intestine. Collectively, our study demonstrates the impact of ALA on the gut microbiome and reveals the nutritional regulation of gut morphology.

Parecoxib does not suppress thromboxane synthesis in newborn piglets with group B streptococcal sepsis.

Group B streptococci (GBS) cause fatal sepsis in newborns. Strong activation of thromboxane synthesis is assumed to correlate with severe pulmonary hypertension. In this study we compared the impact of indomethacin versus parecoxib on hemodynamics and outcome and investigated the pharmacological effects on thromboxane synthesis and EP-3 receptor gene expression. Whereas both parecoxib and indometacin reduced expression of thromboxane synthase and EP-3 receptor in infected lung tissue, parecoxib did not suppress urine levels of thromboxane like indometacin. We presume that COX-2 inhibition in GBS sepsis is associated with enhanced thrombogenicity.

Stability of prostaglandin E(2) (PGE (2)) embedded in poly-D,L: -lactide-co-glycolide microspheres: a pre-conditioning approach for tissue engineering applications.

Prostaglandin E(2) (PGE(2)) is involved in angiogenesis, bone repair and cartilage metabolism. Thus, PGE(2) might represent a suitable signaling molecule in different tissue engineering applications. PGE(2) also has a short half-life time. Its incorporation into poly-D: ,L: -lactide-co-glycolide (PLGA) microspheres was demonstrated in a previous study. However, the stability of bioactive PGE(2) in these microspheres is unknown. With an adjusted mass spectrometry assay we investigated the amount of incorporated PGE(2) and the stability of PGE(2) in conventional cell culture medium and in PLGA microspheres. The stability of PGE(2) was closely pH dependent. Strong acidic or basic environments reduced the half-life from 300 h (pH 2.6-4.0) to below 50 h at pH 2.0 or pH 8.8. The half-life of PGE(2) incorporated into poly-D: ,L: -lactide-co-glycolide increased drastically to 70 days at 37 degrees C and to 300 days at 8 degrees C. Analysis with scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated a distinct nanostructure of the polymeric phase and both nano- and microporosity.

Determination of chamazulene carboxylic acid in serum by high-performance liquid chromatography. Development and validation.

A new, simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of chamazulene carboxylic acid (CCA) in serum. The technique is based on a single liquid-liquid extraction of the substance using ibuprofen as internal standard (I.S.). The separation was achieved on a C(18) reversed-phase column using acetonitrile/water (4:6, pH 3) as mobile phase. The effluent was monitored at 221 and 286 nm. The calibration curves were linear over the concentration range of 0.1-30 microg/ml. The intra- and inter-day RSDs were in all cases less than 15 and 11%, respectively. The limit of quantitation was 0.1 microg/ml. The assay was developed and validated to be applied in a pharmacokinetic study in healthy volunteers.

Determination of misoprostol free acid in human breast milk and serum by gas chromatography/negative ion chemical ionization tandem mass spectrometry.

To study an expected transition of misoprostol from human blood into breast milk, a novel method for the determination of its active metabolite misoprostol acid (MPA) was developed. MPA was determined in serum and breast milk samples by an isotope dilution assay using gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS). After addition of (15S)-15-methylprostaglandin E(2) (15-methyl-PGE(2)) as an internal standard, MPA was extracted from both matrices using a reversed-phase cartridge. The prostanoids were derivatized with O-2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride (PFBHA) and 2,3,4,5,6-pentafluorobenzyl bromide (PFBB) to the pentafluorobenzyl oxime (PFBO)-pentafluorobenzyl ester (PFB) derivatives. The sample was subjected to thin-layer chromatography with ethyl acetate-hexane (1 : 1 (v/v)) as the developing solvent. The corresponding zone was extracted. After derivatization to the trimethylsilyl ether, MPA was determined by GC/NICI-MS/MS using the [molecule (M) - pentafluorobenzyl (PFB)](-) ([P](-)) ions as precursor in the negative ion chemical ionization mode. The product ions used for quantification were [P - 2TMSOH - C(6)F(5)CH(2)OH](-) (MPA) and [P - 2TMSOH - C(6)F(5)CH(2)OH - CO(2)](-)(15-methyl-PGE(2)), respectively. The limit of quantification for MPA was approximately 1 pg ml(-1) in breast milk and serum samples. The correlation coefficients of the calibration curves for MPA were r > 0.997 in the 0.5-2000 pg ml(-1) range for both tested matrices.

The pharmacokinetics of the prostaglandin E1 analogue misoprostol in plasma and colostrum after postpartum oral administration.

OBJECTIVE: To study pharmacokinetics of prostaglandin E1 analogue, misoprostol in plasma and colostrum after postpartum oral administration. STUDY DESIGN: Twenty women received 600 microg doses of misoprostol orally after delivery. Plasma levels of the principal metabolite, misoprostol acid, were measured at 2, 10, 20, 30, 40, 50, 60, 90, 120, 180, 240 and 300 min (48 samples). Colostrum was expressed from the breasts to measure misoprostol acid at 60, 120, 180, 240, and 300 min (24 samples). Assay was done using isotope dilution gas chromatography (GC)/negative ion chemical ionisation mass spectrometry (MS). RESULTS: The plasma concentration of misoprostol acid rose quickly. Two minutes after oral administration its mean level was 91.5 pg/ml, peaked at 20 min (344 pg/ml), then fell steeply by 120 min (27.8 pg/ml) and remained low for the duration of the study. Misoprostol acid in colostrum reached maximum concentration of 20.9 pg/m within 1h after oral administration. It then declined gradually to 17.8 pg/ml at 2h, 2.8 pg/ml at 4h and to <1 pg/ml at 5h. Areas under misoprostol concentration versus time curves up to 5h were 290.1 pgh/ml in the plasma and 51.4 pgh/ml in colostrum, respectively. CONCLUSION: Misoprostol acid is secreted in colostrum within 1h of oral administration of 600 microg of misoprostol; the pharmacokinetics of misoprostol after oral administration during postpartum is similar to that of other pregnancy periods.

Phenotypic redifferentiation and cell cluster formation of cultured human articular chondrocytes in a three-dimensional oriented gelatin scaffold in the presence of PGE2--first results of a pilot study.

Modern tissue engineering strategies comprise three elemental parameters: cells, scaffolds and growth factors. Articular cartilage represents a highly specialized tissue which allows frictionless gliding of corresponding articulating surfaces. As the regenerative potential of cartilage is low, tissue engineering-based strategies for cartilage regeneration represent a huge challenge. Prostaglandins function as regulators in cartilage development and metabolism, especially in growth plate chondrocytes. In this study, it was analyzed if prostaglandin E2 (PGE2 ) has an effect on the phenotypic differentiation of human chondrocytes cultured in a three-dimensional (3D) gelatin-based scaffold made by directional freezing and subsequent freeze-drying. As a result, it was clearly demonstrated that low doses of PGE2 revealed beneficial effects on the phenotypic differentiation and collagen II expression of human articular chondrocytes in this 3D cell culture system. In conclusion, PGE2 is an interesting candidate for tissue engineering applications since it represents an already well-studied molecule which is available in pharmaceutical quality.

Ozone induces synthesis of systemic prostacyclin by cyclooxygenase-2 dependent mechanism in vivo.

Under certain pathological conditions, e.g., infectious or neoplastic diseases, application of ozone exerts therapeutic effects. However, pharmacological mechanisms are not understood. Since an interaction with the arachidonic acid metabolism is suggested we investigated the effect of intraperitoneal insufflation of ozone on prostanoid system in vivo. Upon ozone application (4 mg/kg) to rats we observed an approximate 3-fold increase in excretion rate of 6-keto-prostaglandin (PG) F1α and of 2,3-dinor-6-keto-PG F1α, the measurable stable products of prostacyclin. In plasma and vessel tissue 6-keto-PG F1α concentration was also significantly increased. In contrast, excretion rates for PGE2 and thromboxane (TX) B2 did not change. F2-isoprostanes, regarded as endogenous indicators of oxidative stress, were also unaffected by ozone application. Oxygen insufflation used as control was without any effect on prostanoid levels. Ozone caused increase in 6-keto-PG F1α by arterial but not by venous vessel tissues with peak activity 6-9h following insufflation. The increase in PGI2 synthesis was dependent on cyclooxygenase (COX)-2 activity, demonstrated by its sensitivity towards COX-2 inhibition, and by enhanced COX-2 mRNA and protein expression in vessels. Ozone exerted no rise in excretion rate of prostacyclin metabolites in COX-2(-/-) but in COX-1(-/-) mice. Enzymatic activity and mRNA expression of vascular PGI2 synthase (PGIS) was unaffected by ozone treatment. In summary our study shows for the first time that ozone insufflation causes enhanced expression of COX-2 in the vessel system leading to exclusive elevation of systemic PGI2 levels. We assume that PGI2 stimulation may contribute to the beneficial effects of ozone treatment.

The effect of the pro-inflammatory cytokine tumor necrosis factor-alpha on human joint capsule myofibroblasts.

INTRODUCTION: Previous studies have shown that the number of myoblastically differentiated fibroblasts known as myofibroblasts (MFs) is significantly increased in stiff joint capsules, indicating their crucial role in the pathogenesis of post-traumatic joint stiffness. Although the mode of MFs' function has been well defined for different diseases associated with tissue fibrosis, the underlying mechanisms of their regulation in the pathogenesis of post-traumatic joint capsule contracture are largely unknown. METHODS: In this study, we examined the impact of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on cellular functions of human joint capsule MFs. MFs were challenged with different concentrations of TNF-alpha with or without both its specifically inactivating antibody infliximab (IFX) and cyclooxygenase-2 (COX2) inhibitor diclofenac. Cell proliferation, gene expression of both alpha-smooth muscle actin (alpha-SMA) and collagen type I, the synthesis of prostaglandin derivates E2, F1A, and F2A, as well as the ability to contract the extracellular matrix were assayed in monolayers and in a three-dimensional collagen gel contraction model. The alpha-SMA and COX2 protein expressions were evaluated by immunofluorescence staining and Western blot analysis. RESULTS: The results indicate that TNF-alpha promotes cell viability and proliferation of MFs, but significantly inhibits the contraction of the extracellular matrix in a dose-dependent manner. This effect was associated with downregulation of alpha-SMA and collagen type I by TNF-alpha application. Furthermore, we found a significant time-dependent upregulation of prostaglandin E2 synthesis upon TNF-alpha treatment. The effect of TNF-alpha on COX2-positive MFs could be specifically prevented by IFX and partially reduced by the COX2 inhibitor diclofenac. CONCLUSIONS: Our results provide evidence that TNF-alpha specifically modulates the function of MFs through regulation of prostaglandin E2 synthesis and therefore may play a crucial role in the pathogenesis of joint capsule contractures.

Immobilization and controlled release of prostaglandin E2 from poly-L-lactide-co-glycolide microspheres.

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite involved in physiological homeostasis and numerous pathophysiological conditions. Furthermore, it has been demonstrated that prostaglandins have a stimulating effect not only on angiogenesis in situ and in vitro but also on chondrocyte proliferation in vitro. Thus, PGE(2) represents an interesting signaling molecule for various tissue engineering strategies. However, under physiological conditions, PGE(2) has a half-life time of only 10 min, which limits its use in biomedical applications. In the present study, we investigated if the incorporation of PGE(2) into biodegradable poly-L-lactide-co-glycolide microspheres results in a prolonged release of this molecule in its active form. PGE(2)-modified microspheres were produced by a cosolvent emulsification method using CHCl(3) and HFIP as organic solvents and PVA as emulsifier. Thirteen identical batches were produced; and to each batch 1.0 mL of serum-free medium was added. The medium was removed at defined time points and then analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) to measure the residual PGE(2) content. In this study we demonstrated the prolonged release of PGE(2), showing a linear increase over the first 12 h, followed by a plateau and a slow decrease. The microspheres were further characterized by scanning electron microscopy.

Dopamides, vanillylamides, ethanolamides, and arachidonic acid amides of anti-inflammatory and analgesic drug substances as TRPV1 ligands.

Drug substances can be acylated metabolically to give derivatives with specific and strong molecular effects. We generated potentially naturally occurring acid amides of several anti-inflammatory and analgesic drugs. In the amides, the drug moieties served either as amine or acid components. All compounds were evaluated for activity toward transient receptor potential vanilloid subfamily member 1 (TRPV1) in a cell-based Ca2+ influx assay; TRPV1 is a key receptor in the pain pathway and a promising target for analgesic drugs. We found that dopamine amides of fenamic acids have TRPV1 agonist activity in the nanomolar range, and that the arachidonoyl amide of a dipyrone metabolite has TRPV1 antagonist activity. Flufenamic acid dopamide, the most potent TRPV1 agonist reported herein, retains the cyclooxygenase (COX) inhibition properties of the parent compound flufenamic acid. Thus it acts on two different major players in the pain processing machinery. The compounds could be further keys to understanding the mechanism of action of fenamates and dipyrone at the molecular level. The fenamic acid dopamine amides qualify as new lead structures for drug development.

New analgesics synthetically derived from the paracetamol metabolite N-(4-hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide.

N-(4-hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide (AM404) is a metabolite of the well-known analgesic paracetamol. AM404 inhibits endocannabinoid cellular uptake, binds weakly to CB1 and CB2 cannabinoid receptors, and is formed by fatty acid amide hydrolase (FAAH) in vivo. We prepared three derivatives of this new (endo)cannabinoid using bioisosteric replacement (1), homology (2), and derivatization (3) of the 4-aminophenol moiety in AM404 and tested them against CB1, CB2, and FAAH. We found affinities toward both cannabinoid receptors equal to or greater than that of AM404. Shortening the acyl chain from C20 to C2 led to three new paracetamol analogues: N-(1H-indazol-5-yl)acetamide (5), N-(4-hydroxybenzyl)acetamide (6), and N-(4-hydroxy-3-methoxyphenyl)acetamide (7). Again, 5, 6, and 7 were tested against CB1, CB2, and FAAH without significant activity. However, 5 and 7 behaved like inhibitors of cyclooxygenases in whole blood assays. Finally, 5 (50 mg/kg) and 6 (275 mg/kg) displayed analgesic activities comparable to paracetamol (200 mg/kg) in the mouse formalin test.