Conjugation of peptides to short-acyl-chain ceramides for delivery across mucosal cell barriers.

Robust transport of therapeutic peptides and other medicinal molecules across tight epithelial barriers would overcome the major obstacle to oral delivery. We have already demonstrated that peptides conjugated to gangliosides (GM1 and GM3) having non-native short N-acyl groups hijack the endogenous process of intracellular lipid sorting resulting in transcytosis and delivery across epithelial barriers in vitro and in vivo. Here, we report synthetic methodologies to covalently conjugate peptides directly to short-acyl-chain C6-ceramides. We found that the short-acyl-chain ceramide domain is solely responsible for transcytosis in vitro. This clarifies and expands the platform of short-acyl-chain sphingolipids for conjugated peptide delivery across tight mucosal cell barriers from gangliosides to just the ceramide itself.

Metabolic studies of synaptamide in an immortalized dopaminergic cell line.

INTRODUCTION: Synaptamide, the N-acylethanolamine of docosahexaenoic acid (DHA), is structurally similar to the endocannabinoid N-arachidonoylethanolamine, anandamide. It is an endogenous ligand at the orphan G-protein coupled receptor 110 (GPR110; ADGRF1), and induces neuritogenesis and synaptogenesis in hippocampal and cortical neurons, as well as neuronal differentiation in neural stem cells. PURPOSE: Our goal was to characterize the metabolic fate (synthesis and metabolism) of synaptamide in a dopaminergic cell line using immortalized fetal mesencephalic cells (N27 cells). Both undifferentiated and differentiating N27 cells were used in this study in an effort to understand synaptamide synthesis and metabolism in developing and adult cells. METHODS: Radiotracer uptake and hydrolysis assays were conducted in N27 cells incubated with [1-14C]DHA or with one of two radioisotopomers of synaptamide: [α,ß-14C2]synaptamide and [1-14C-DHA]synaptamide. RESULTS: Neither differentiated nor undifferentiated N27 cells synthesized synaptamide from radioactive DHA, but both rapidly incorporated radioactivity from exogenous synaptamide into membrane phospholipids, regardless of which isotopomer was used. Pharmacological inhibition of fatty acid amide hydrolase (FAAH) reduced formation of labeled phospholipids in undifferentiated but not differentiated cells. CONCLUSIONS: In undifferentiated cells, synaptamide uptake and metabolism is driven by its enzymatic hydrolysis (fatty acid amide hydrolase; FAAH), but in differentiating cells, the process seems to be FAAH independent. We conclude that differentiated and undifferentiated N27 cells utilize synaptamide via different mechanisms. This observation could be extrapolated to how different mechanisms may be in place for synaptamide uptake and metabolism in developing and adult dopaminergic cells.

Stereochemical Structure Activity Relationship Studies (S-SAR) of Tetrahydrolipstatin.

Tetrahydrolipstatin (THL), its enantiomer, and an additional six diastereomers were evaluated as inhibitors of the hydrolysis of p-nitrophenyl butyrate by porcine pancreatic lipase. IC50s were found for all eight stereoisomers ranging from a low of 4.0 nM for THL to a high of 930 nM for the diastereomer with the inverted stereocenters at the 2,3,2'-positions. While the enantiomer of THL was also significantly less active (77 nM) the remaining five stereoisomers retained significant inhibitory activities (IC50s = 8.0 to 20 nM). All eight compounds were also evaluated against three human cancer cell lines (human breast cancers MCF-7 and MDA-MB-231, human large-cell lung carcinoma H460). No appreciable cytotoxicity was observed for THL and its seven diastereomers, as their IC50s in a MTT cytotoxicity assay were all greater than 3 orders of magnitude of camptothecin.

N-Docosahexaenoylethanolamine (synaptamide): Carbon-14 radiolabeling and metabolic studies.

N-Docosahexaenoylethanolamine (synaptamide) is structurally similar to the endocannabinoid N-arachidonoylethanolamine (anandamide), but incorporates the omega-3 22:6 fatty acid docosahexaenoic acid (DHA) in place of the omega-6 20:4 fatty acid arachidonic acid (AA). Some brain membrane lipid effects may be mediated via synaptamide. In competition experiments with mouse brain homogenate in vitro, we found that synaptamide was an order-of-magnitude poorer inhibitor of radioactive anandamide hydrolysis than was anandamide itself. Also, enzyme-mediated hydrolysis of synaptamide was observed to occur at a slower rate than for anandamide. We have synthesized synaptamide radiolabeled with carbon-14 in both the ethanolamine ([α,ß-14C2]synaptamide) and in the DHA ([1-14C]synaptamide) moieties. The brain penetration, distribution, and metabolism of radiolabeled synaptamide were studied in mice in vivo relative to anandamide, DHA, and AA. Brain uptake of labeled synaptamide was greater than for labeled DHA, consistent with previous studies of labeled anandamide and AA in our laboratory. After administering either isotopomer of radiolabeled synaptamide, radiolabeled phospholipids were found in mouse brain. Pretreatment of mice with PF3845, a potent, specific inhibitor of fatty acid amide hydrolase (FAAH), eliminated formation of labeled phospholipids measured after 15min, suggesting that synaptamide is hydrolyzed nearly exclusively by FAAH, though it is a poorer substrate for FAAH than anandamide.

Brain uptake and metabolism of the endocannabinoid anandamide labeled in either the arachidonoyl or ethanolamine moiety.

INTRODUCTION: Anandamide (N-arachidonoylethanolamine) is a retrograde neuromodulator that activates cannabinoid receptors. The concentration of anandamide in the brain is controlled by fatty acid amide hydrolase (FAAH), which has been the focus of recent drug discovery efforts. Previous studies in C57BL/6 mice using [3H-arachidonoyl]anandamide demonstrated deposition of tritium in thalamus and cortical areas that was blocked by treatment with an FAAH inhibitor and that was not seen in FAAH-knockout mice. This suggested that long chain fatty acid amides radiolabeled in the fatty acid moiety might be useful as ex vivo and in vivo radiotracers for FAAH, since labeled fatty acid released by hydrolysis would be rapidly incorporated into phospholipids with long metabolic turnover periods. METHODS: Radiotracers were administered intravenously to conscious Swiss-Webster mice, and radioactivity concentrations in brain areas was quantified and radiolabeled metabolites determined by radiochromatography. RESULTS: [14C]Arachidonic acid, [14C-arachidonoyl]anandamide and [14C-ethanolamine]anandamide, and also [14C]myristic acid, [14C-myristoyl]myristoylethanolamine and [14C-ethanolamine]myristoyl-ethanolamine all had very similar distribution patterns, with whole brain radioactivity concentrations of 2-4% injected dose per gram. Pretreatment with the potent selective FAAH inhibitor URB597 did not significantly alter distribution patterns although radiochromatography demonstrated that the rate of incorporation of label from [14C]anandamide into phospholipids was decreased. Pretreatment with the muscarinic agonist arecoline which increases cerebral perfusion increased brain uptake of radiolabel from [14C]arachidonic acid and [14C-ethanolamine]anandamide, and (in dual isotope studies) from the unrelated tracer [125I]RTI-55. CONCLUSIONS: Together with our previously published study with [18F-palmitoyl]16-fluoro-palmitoylethanolamine, the data show that the primary determinant of brain uptake for these tracers in Swiss-Webster mice is initial distribution according to blood flow. It is possible that recently reported strain differences in long chain fatty acid trafficking between C57BL/6 and Swiss-Webster mice are responsible for the differences between our results using [14C]anandamide and the earlier studies using [3H]anandamide.

Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases.

Identifying an enzyme's substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.

17ß-estradiol (E2) in membranes: Orientation and dynamic properties.

Non-genomic membrane effects of estrogens are of great interest because of the diverse biological activities they may elicit. To further our understanding of the molecular features of the interaction between estrogenic hormones and membrane bilayers, we have determined the preferred orientation, location, and dynamic properties of 17ß-estradiol (E2) in two different phospholipid membrane environments using (2)H-NMR and 2D (1)H-(13)C HSQC in conjunction with molecular dynamics simulations. Unequivocal spectral assignments to specific (2)H labels were made possible by synthesizing six selectively deuterated E2 molecules. The data allow us to conclude that the E2 molecule adopts a nearly "horizontal" orientation in the membrane bilayer with its long axis essentially perpendicular to the lipid acyl-chains. All four rings of the E2 molecule are located near the membrane interface, allowing both the E2 3-OH and the 17ß-OH groups to engage in hydrogen bonding and electrostatic interactions with polar phospholipid groups. The findings augment our knowledge of the molecular interactions between E2 and membrane bilayer and highlight the asymmetric nature of the dynamic motions of the rigid E2 molecule in a membrane environment.

Synthesis and characterization of Se-adenosyl-L-selenohomocysteine selenoxide.

Selenium is an essential micronutrient in humans due to the important roles of the selenocysteine-containing selenoproteins. Organoselenium metabolites are generally found to be substrates for the biochemical pathways of their sulfur analogs, and the redox chemistry of selenomethionine and some other metabolites have been previously reported. We now report the first synthesis and characterization of Se-adenosylselenohomocysteine selenoxide (SeAHO) prepared via hydrogen peroxide oxidation of Se-adenosylselenohomocysteine (SeAH). The selenoxide SeAHO, in contrast to its corresponding sulfoxide S-adenosylhomocysteine (SAHO), can form hydrate, has an electrostatic interaction between the α-amino acid moiety and the highly polar selenoxide functional group, and readily oxidizes glutathione (GSH) and cysteine thiols.

Integrity of (111)In-radiolabeled superparamagnetic iron oxide nanoparticles in the mouse.

INTRODUCTION: Iron-oxide nanoparticles can act as contrast agents in magnetic resonance imaging (MRI), while radiolabeling the same platform with nuclear medicine isotopes allows imaging with positron emission tomography (PET) or single-photon emission computed tomography (SPECT), modalities that offer better quantification. For successful translation of these multifunctional imaging platforms to clinical use, it is imperative to evaluate the degree to which the association between radioactive label and iron oxide core remains intact in vivo. METHODS: We prepared iron oxide nanoparticles stabilized by oleic acid and phospholipids which were further radiolabeled with (59)Fe, (14)C-oleic acid, and (111)In. RESULTS: Mouse biodistributions showed (111)In preferentially localized in reticuloendothelial organs, liver, spleen and bone. However, there were greater levels of (59)Fe than (111)In in liver and spleen, but lower levels of (14)C. CONCLUSIONS: While there is some degree of dissociation between the (111)In labeled component of the nanoparticle and the iron oxide core, there is extensive dissociation of the oleic acid component.

Synthesis and preliminary evaluation of N-(16-18F-fluorohexadecanoyl)ethanolamine (18F-FHEA) as a PET probe of N-acylethanolamine metabolism in mouse brain.

N-Acylethanolamines are lipid signaling molecules found throughout the plant and animal kingdoms. The best-known mammalian compound of this class is anandamide, N-arachidonoylethanolamine, one of the endogenous ligands of cannabinoid CB1 and CB2 receptors. Signaling by N-acylethanolamines is terminated by release of the ethanolamine moiety by hydrolyzing enzymes such as fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing amidase (NAAA). Herein, we report the design and synthesis of N-(16-(18)F-fluorohexadecanoyl)ethanolamine ((18)F-FHEA) as a positron emission tomography (PET) probe for imaging the activity of N-acylethanolamine hydrolyzing enzymes in the brain. Following intravenous administration of (18)F-FHEA in Swiss Webster mice, (18)F-FHEA was extracted from blood by the brain and underwent hydrolysis at the amide bond and incorporation of the resultant (18)F-fluorofatty acid into complex lipid pools. Pretreatment of mice with the FAAH inhibitor URB-597 (1 mg/kg IP) resulted in significantly slower (18)F-FHEA incorporation into lipid pools, but overall (18)F concentrations in brain regions were not altered. Likewise, pretreatment with a NAAA inhibitor, (S)-N-(2-oxo-3-oxytanyl)biphenyl-4-carboxamide (30 mg/kg IV), did not significantly affect the uptake of (18)F-FHEA in the brain. Although evidence was found that (18)F-FHEA behaves as a substrate of FAAH in the brain, the lack of sensitivity of brain uptake kinetics to FAAH inhibition discourages its use as a metabolically trapped PET probe of N-acylethanolamine hydrolyzing enzyme activity.

Assay and inhibition of diacylglycerol lipase activity.

A series of N-formyl-α-amino acid esters of ß-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.

A methodology for radiolabeling of the endocannabinoid 2-arachidonoylglycerol (2-AG).

The metabolic intermediate and endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) has not been readily labeled, primarily because of its instability toward rearrangement. We now detail a synthetic method that easily gives tritiated 2-AG from [5,6,8,9,11,12,14,15-(3)H(N)]arachidonic acid in two steps. We utilized a short chain 1,3-diacylglycerol and proceeded through the "structured lipid" [5'',6'',8'',9'',11'',12'',14'',15''-(3)H(N)]2-arachidonoyl-1,3-dibutyrylglycerol, a triacylglycerol that was conveniently deprotected in ethanol with acrylic beads containing Candida antarctica lipase B to give [5'',6'',8'',9'',11'',12'',14'',15''-(3)H(N)]2-arachidonoylglycerol ([(3)H]2-AG). The flash chromatographic separation necessary to isolate the labeled 2-acylglycerol [(3)H]2-AG resulted in only 4% of the rearrangement byproducts that have been a particular problem with previous methodologies. This reliable "kit" method to prepare the radiolabeled endocannabinoid as needed gave tritiated 2-arachidonoylglycerol [(3)H]2-AG with a specific activity of 200 Ci/mmol for enzyme assays, metabolic studies, and tissue imaging. It has been run on unlabeled materials on over 10 mg scales and should be generally applicable to other 2-acylglycerols.

The Conformations of 17ß-Estradiol (E2) and 17α-Estradiol as Determined by Solution NMR.

The conformational structures of the hormone 17ß-estradiol (E2) and the epimeric 17α-estradiol determined by solution NMR spectroscopy and restrained molecular dynamics calculations found a single low energy conformation.

The total synthesis of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1'-(13)C3 and -2,1'-(13)C2 by a novel chemoenzymatic method.

2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine was synthesized with carbon-13 enrichment of the three glycerol carbons and the carbonyl of the stearoyl group. Phospholipase A(2) was utilized to give optically pure lyso-PC, and only 3% acyl migration occurred during reacylation with arachidonic acid anhydride. This phospholipid is an important biosynthetic precursor of arachidonic acid metabolites as well as the endocannabinoid 2-arachidonoylglycerol (2-AG), and is now available for NMR studies.

The preparation of 2-O-[1'-C]arachidonoyl-1-O-stearoyl-sn-glycerol.

2-O-Arachidonoyl-1-O-stearoyl-sn-glycerol is the most abundant molecular species of the 1,2-diacyl-sn-glycerol signaling lipids in neural tissue. The facile preparation of 2-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycerol from 2-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine at a hexane and phosphate buffer interface with phospholipase C was demonstrated on a 20 µCi scale in 83% radiochemical yield. The specific activity of the product 2-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycerol was 57.0 mCi/mmol and the radiochemical purity was determined to be >99% by TLC. The hydrolysis of this lipid biosynthetic intermediate with lipoprotein lipase was shown to produce 2-O-[1'-(14)C]arachidonoylglycerol (2-AG). The (14)C-radiolabeled monoacylglycerol 2-AG is an endogenous cannabinoid receptor-signaling molecule (endocannabinoid).

Open vessel mode microwave-assisted synthesis of 2-oxazolines from carboxylic acids.

Microwave-assisted synthesis of 2-oxazolines from carboxylic acids using the open vessel technique is described. This efficient method involves direct condensation of carboxylic acids with excess 2-amino-2-methyl-1-propanol at 170 °C to give the corresponding 2-oxazolines in moderate to excellent yields.

Bornyl- and isobornyl-Delta8-tetrahydrocannabinols: a novel class of cannabinergic ligands.

Structure-activity relationship studies of classical cannabinoid analogues have established that the C3 aliphatic side chain plays a pivotal role in determining cannabinergic potency. In earlier work, we provided evidence for the presence of subsites within the CB1 and CB2 cannabinoid receptor binding domains that can accommodate bulky conformationally defined substituents at the C3 alkyl side chain pharmacophore of classical cannabinoids. We have now extended this work with the synthesis of a series of Delta (8)-THC analogues in which bornyl substituents are introduced at the C3 position. Our results indicate that, for optimal interactions with both CB1 and CB2 receptors, the bornyl substituents need to be within close proximity of the tricyclic core of Delta (8)-THC and that the conformational space occupied by the C3 substituents influences CB1/CB2 receptor subtype selectivity.

Synthesis and characterization of 2-substituted bornane pharmacophores for novel cannabinergic ligands.

Analogously to the fenchyl and adamantyl groups, the bornyl and epimeric isobornyl groups are compact lipophilic substituents that can be incorporated into drug design to improve pharmacological or physicochemical properties. Methods are reported for the synthesis and characterization of 2-substituted norbornanes and bornanes that can serve as novel cannabinergic ligand intermediates.

Cannabilactones: a novel class of CB2 selective agonists with peripheral analgesic activity.

The identification of the CB2 cannabinoid receptor has provided a novel target for the development of therapeutically useful cannabinergic molecules. We have synthesized benzo[ c]chromen-6-one analogs possessing high affinity and selectivity for this receptor. These novel compounds are structurally related to cannabinol (6,6,9-trimethyl-3-pentyl-6 H-benzo[ c]chromen-1-ol), a natural constituent of cannabis with modest CB2 selectivity. Key pharmacophoric features of the new selective agonists include a 3-(1',1'-dimethylheptyl) side chain and a 6-oxo group on the cannabinoid tricyclic structure that characterizes this class of compounds as "cannabilactones." Our results suggest that the six-membered lactone pharmacophore is critical for CB2 receptor selectivity. Optimal receptor subtype selectivity of 490-fold and subnanomolar affinity for the CB2 receptor is exhibited by a 9-hydroxyl analog 5 (AM1714), while the 9-methoxy analog 4b (AM1710) had a 54-fold CB2 selectivity. X-ray crystallography and molecular modeling show the cannabilactones to have a planar ring conformation. In vitro testing revealed that the novel compounds are CB2 agonists, while in vivo testing of cannabilactones 4b and 5 found them to possess potent peripheral analgesic activity.

Conformationally constrained analogues of 2-arachidonoylglycerol.

Novel monocyclic analogues of 2-arachidonoylglycerol (2-AG) were designed in order to explore the pharmacophoric conformations of this endocannabinoid ligand at the key cannabinergic proteins. All 2-arachidonoyl esters of 1,2,3-cyclohexanetriol [meso-7 (AM5504), (+/-)-8 (AM5503), and meso-9 (AM5505)] were synthesized by regioselective acylation of 2,3-dihydroxycyclohexanone followed by selective reductions. The optically active isomers (+)-8 (AM4434) and (-)-8 (AM4435) were synthesized from (2S,3S)- and (2R,3R)-2,3-dihydroxycyclohexanone, respectively, via a chemoenzymatic route. These head group constrained and conformationally restricted analogues of 2-AG as well as the 1-keto precursors were evaluated as substrates for the endocannabinoid deactivating hydrolytic enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), and also were tested for their affinities for CB1 and CB2 cannabinoid receptors. The observed biochemical differences between these ligands can help define the conformational requirements for 2-AG activity at each of the above endocannabinoid protein targets.