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1.  The Interaction of Fatty Acid Amide Hydrolase (FAAH) Inhibitors with an Anandamide Carrier Protein Using 19F-NMR 
The AAPS Journal  2013;15(2):477-482.
It has been reported that the endocannabinoid anandamide (AEA) binds to a class of fatty acid-binding proteins and serum albumin which can serve as carrier proteins and potentiate the cellular uptake of AEA and its intracellular translocation. Here, we employed 19F nuclear magnetic resonance spectroscopy to study the interactions of serum albumin with two inhibitors of fatty acid amide hydrolase (FAAH), the enzyme involved in the deactivation of anandamide. We found that, for both inhibitors AM5206 and AM5207, the primary binding site on serum albumin is drug site 1 located at subdomain IIA. Neither inhibitor binds to drug site 2. While AM5207 binds exclusively to drug site 1, AM5206 also interacts with other fatty acid-binding sites on serum albumin. Additionally, AM5206 has an affinity for serum albumin approximately one order of magnitude higher than that of AM5207. The data suggest that interactions of FAAH inhibitors with albumin may provide added advantages for their ability to modulate endocannabinoid levels for a range of applications including analgesia, antiemesis, and neuroprotection.
doi:10.1208/s12248-013-9455-9
PMCID: PMC3675727  PMID: 23344792
anandamide carrier proteins; FAAH; 19F-NMR; serum albumin
2.  Human Cannabinoid 1 GPCR C-Terminal Domain Interacts with Bilayer Phospholipids to Modulate the Structure of its Membrane Environment 
The AAPS Journal  2011;13(1):92-98.
G protein-coupled receptors (GPCRs) play critical physiological and therapeutic roles. The human cannabinoid 1 GPCR (hCB1) is a prime pharmacotherapeutic target for addiction and cardiometabolic disease. Our prior biophysical studies on the structural biology of a synthetic peptide representing the functionally significant hCB1 transmembrane helix 7 (TMH7) and its cytoplasmic extension, helix 8 (H8), [hCB1(TMH7/H8)] demonstrated that the helices are oriented virtually perpendicular to each other in membrane-mimetic environments. We identified several hCB1(TMH7/H8) structure-function determinants, including multiple electrostatic amino-acid interactions and a proline kink involving the highly conserved NPXXY motif. In phospholipid bicelles, TMH7 structure, orientation, and topology relative to H8 are dynamically modulated by the surrounding membrane phospholipid bilayer. These data provide a contextual basis for the present solid-state NMR study to investigate whether intermolecular interactions between hCB1(TMH7/H8) and its phospholipid environment may affect membrane-bilayer structure. For this purpose, we measured 1H–13C heteronuclear dipolar couplings for the choline, glycerol, and acyl-chain regions of dimyristoylphosphocholine in a magnetically aligned hCB1(TMH7/H8) bicelle sample. The results identify discrete regional interactions between hCB1(TMH7/H8) and membrane lipid molecules that increase phospholipid motion and decrease phospholipid order, indicating that the peptide’s partial traversal of the bilayer alters membrane structure. These data offer new insight into hCB1(TMH7/H8) properties and support the concept that the membrane bilayer itself may serve as a mechanochemical mediator of hCB1/GPCR signal transduction. Since interaction with its membrane environment has been implicated in hCB1 function and its modulation by small-molecule therapeutics, our work should help inform hCB1 pharmacology and the design of hCB1-targeted drugs.
doi:10.1208/s12248-010-9244-7
PMCID: PMC3032089  PMID: 21234731
conformational switch; integral-membrane protein topology; molecular hinge; NMR; signal transduction
3.  Antinociceptive Effects of Racemic AM1241 and Its Chirally Synthesized Enantiomers: Lack of Dependence upon Opioid Receptor Activation 
The AAPS Journal  2010;12(2):147-157.
Cannabinoid CB2 receptors represent a therapeutic target that circumvents unwanted central side effects (e.g., psychoactivity and/or addiction) associated with activation of CB1 receptors. One of the primary investigative tools used to study functions of the CB2 receptor is the aminoalkylindole (R,S)-AM1241. However, (R,S)-AM1241 has been described as an atypical CB2 agonist which produces antinociception mediated indirectly by opioid receptors. (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, were evaluated for antinociception in response to thermal (Hargreaves) and mechanical (von Frey) stimulation. Pharmacological specificity was established using antagonists for CB1 (rimonabant [SR141716]) and CB2 (SR144528). The opioid antagonist naloxone was administered locally in the paw or systemically to evaluate the contribution of opioid receptors to CB2-mediated antinociception produced by (R,S)-AM1241, (R)-AM1241, and (S)-AM1241. Comparisons were made with the opioid analgesic morphine. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 (0.033–10 mg/kg i.p.) produced antinociception to thermal, but not mechanical, stimulation of the hindpaw in naive rats. Antinociception produced by (R,S)-AM1241 and (S)-AM1241 exhibited an inverted U-shaped dose response curve. (R)-AM1241 produced greater antinociception than either (S)-AM1241 or (R,S)-AM1241 at the lowest (0.033 and 0.1 mg/kg i.p.) and highest (10 mg/kg i.p.) doses. Similar levels of antinociception were observed at intermediate doses. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 each produced CB2-mediated antinociception that was blocked by SR144528 but not by rimonabant. Local and systemic naloxone blocked morphine-induced antinociception but did not block antinociceptive effects of (R,S)-AM1241, (R)-AM1241, or (S)-AM1241. The antinociceptive effects of the CB2-selective cannabinoid (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, are not dependent upon opioid receptors.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-009-9170-8) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-009-9170-8
PMCID: PMC2844508  PMID: 20127295
antinociception; cannabinoid; CB2; endogenous opioid; naloxone; pain
4.  Endocannabinoid metabolomics: A novel liquid chromatography-mass spectrometry reagent for fatty acid analysis 
The AAPS Journal  2006;8(4):E655-E660.
We have synthesized 4,4-dimethoxyoxazoline derivatives of several fatty acids associated with the endocannabinoid metabolome using tris(hydroxymethyl)aminomethane in a 1-step reaction by microwave irradiation. The derivatization incorporates a nitrogen into the final product, which allows for improved detection by liquid chromatographymass spectrometry in positive atmospheric pressure chemical ionization (APCI) mode. Palmitic and oleic acid derivatives show a 200-fold increase in sensitivity compared with the free acids when analyzed in negative-mode APCI. In addition to improving sensitivity, the oxazoline derivatization creates a similar ionization response for the fatty acids tested, which simplifies their quantitation. Fatty acid oxazoline derivatives can be detected using the same conditions optimized for the endocannabinoids, which allows for a simultaneous quantitation of the entire endocannabinoid metabolome.
doi:10.1208/aapsj080474
PMCID: PMC2751361  PMID: 17233529
fatty acids; LC-MS; metabolome; oxazoline derivatives; microwave synthesis
5.  The role of halogen substitution in classical cannabinoids: A CB1 pharmacophore model 
The AAPS Journal  2004;6(4):23-35.
The presence of halogens within the classical cannabinoid structure leads to large variations in the compounds' potencies and affinities for the CB1 receptors. To explore the structure activity relationships within this class of analogs we have used a series of halogen-substituted (−)-Δ8-tetrahydrocannabinol analogs and compared their affinities for the CB1 cannabinoid receptor. Our results indicate that halogen substitution at the end-carbon of the side chain leads to an enhancement in affinity with the bulkier halogens (Br, I) producing the largest effects. Conversely, 2-iodo substitution on the phenolic ring leads to a 2-fold reduction in affinity while iodo-substitution in the C1'-position of the side chain lowers the compound's affinity for CB1 by more than 8-fold. The pharmacophoric requirements resulting from halogen-substitution are explored using computer modeling methods.
doi:10.1208/aapsj060430
PMCID: PMC2751226  PMID: 15760095
tetrahydrocannabinol; halogen substitution; CB1 cannabinoid receptors

Results 1-5 (5)