Apparent CB1 Receptor Rimonabant Affinity Estimates: Combination with THC and Synthetic Cannabinoids in the Mouse In Vivo Triad Model.

Synthetic cannabinoids (SCs) represent an emerging class of abused drugs associated with psychiatric complications and other substantial health risks. These ligands are largely sold over the internet for human consumption, presumably because of their high cannabinoid 1 receptor (CB1R) affinity and their potency in eliciting pharmacological effects similar to Δ9-tetrahydrocannabinol (THC), as well as circumventing laws illegalizing this plant. Factors potentially contributing to the increased prevalence of SC abuse and related hospitalizations, such as increased CB1R efficacy and non-CB1R targets, highlight the need for quantitative pharmacological analyses to determine receptor mediation of the pharmacological effects of cannabinoids. Accordingly, the present study used pA2 and pKB analyses for quantitative determination of CB1R mediation in which we utilized the CB1R-selective inverse agonist/antagonist rimonabant to elicit rightward shifts in the dose-response curves of five SCs (i.e., A-834,735D; WIN55,212-2; CP55,950; JWH-073; and CP47,497) and THC in producing common cannabimimetic effects (i.e., catalepsy, antinociception, and hypothermia). The results revealed overall similarity of pA2 and pKB values for these compounds and suggest that CB1Rs, and not other pharmacological targets, largely mediated the central pharmacological effects of SCs. More generally, affinity estimation offers a powerful pharmacological approach to assess potential receptor heterogeneity subserving in vivo pharmacological effects of SCs.

Stratification of Cannabinoid 1 Receptor (CB1R) Agonist Efficacy: Manipulation of CB1R Density through Use of Transgenic Mice Reveals Congruence between In Vivo and In Vitro Assays.

Synthetic cannabinoids (SCs) are an emerging class of abused drugs that differ from each other and the phytocannabinoid ∆9-tetrahydrocannabinol (THC) in their safety and cannabinoid-1 receptor (CB1R) pharmacology. As efficacy represents a critical parameter to understanding drug action, the present study investigated this metric by assessing in vivo and in vitro actions of THC, two well-characterized SCs (WIN55,212-2 and CP55,940), and three abused SCs (JWH-073, CP47,497, and A-834,735-D) in CB1 (+/+), (+/-), and (-/-) mice. All drugs produced maximal cannabimimetic in vivo effects (catalepsy, hypothermia, antinociception) in CB1 (+/+) mice, but these actions were essentially eliminated in CB1 (-/-) mice, indicating a CB1R mechanism of action. CB1R efficacy was inferred by comparing potencies between CB1 (+/+) and (+/-) mice [+/+ ED50 /+/- ED50], the latter of which has a 50% reduction of CB1Rs (i.e., decreased receptor reserve). Notably, CB1 (+/-) mice displayed profound rightward and downward shifts in the antinociception and hypothermia dose-response curves of low-efficacy compared with high-efficacy cannabinoids. In vitro efficacy, quantified using agonist-stimulated [35S]GTPγS binding in spinal cord tissue, significantly correlated with the relative efficacies of antinociception (r = 0.87) and hypothermia (r = 0.94) in CB1 (+/-) mice relative to CB1 (+/+) mice. Conversely, drug potencies for cataleptic effects did not differ between these genotypes and did not correlate with the in vitro efficacy measure. These results suggest that evaluation of antinociception and hypothermia in CB1 transgenic mice offers a useful in vivo approach to determine CB1R selectivity and efficacy of emerging SCs, which shows strong congruence with in vitro efficacy.

Acute and chronic effects of a contraceptive compound RTI-4587-073(l) on testicular histology and endocrine function in miniature horse stallions.

The objective of this study was to evaluate acute endocrine effects as well as histological changes in testicular parenchyma induced by the contraceptive compound RTI-4587-073(l). Six miniature stallions were used in this experiment. The treatment group (n = 3) received one oral dose of 12.5 mg/kg of RTI-4587-073(l), and the control group (n = 3) received placebo only. The stallions' baseline parameters (semen, testicular dimensions, endocrine values) were collected and recorded for 5 weeks before treatment and for 6 weeks after treatment. Multiple blood samples were collected for endocrine analysis. Testicular biopsies were obtained before treatment, 1 day after treatment and every other week after treatment. Ultrasound exams were performed to monitor the dimensions of the stallions' testes. All stallions were castrated 6 weeks after treatment. Sperm numbers, motility and percentage of morphologically normal sperm decreased (p < 0.05), while the number of immature germ cells increased in ejaculates from treated animals (p < 0.05). Serum concentrations of inhibin and follicle-stimulating hormone did not change. Testosterone concentrations initially transiently decreased (p < 0.05) after administration of RTI-4587-073(l), and increased several days later (p < 0.05). Testicular content of testosterone and estradiol 17-ß was lower in treated stallions than in control stallions on Day 1 after treatment (p < 0.05). Severe disorganization of the seminiferous tubules, significant loss of immature germ cells and complete depletion of elongated spermatids were observed in testicular biopsies obtained from treated stallions 1 day, 2 and 4 weeks after treatment. These changes were still present in the testicular samples taken from treated stallions after castration. The results of this study confirmed that RTI-4587-073(l) has antispermatogenic effects in stallions. Furthermore, we concluded that this compound causes acute sloughing of immature germ cells from the seminiferous tubules. RTI-4587-073(l) has significant but transient effects on Leydig cell function in stallions.

Transcriptional regulation of phospholipid biosynthesis is linked to fatty acid metabolism by an acyl-CoA-binding-protein-dependent mechanism in Saccharomyces cerevisiae.

In the present study, we have used DNA microarray and quantitative real-time PCR analysis to examine the transcriptional changes that occur in response to cellular depletion of the yeast acyl-CoA-binding protein, Acb1p. Depletion of Acb1p resulted in the differential expression of genes encoding proteins involved in fatty acid and phospholipid synthesis (e.g. FAS1, FAS2, ACC1, OLE1, INO1 and OPI3), glycolysis and glycerol metabolism (e.g. GPD1 and TDH1), ion transport and uptake (e.g. ITR1 and HNM1) and stress response (e.g. HSP12, DDR2 and CTT1). In the present study, we show that transcription of the INO1 gene, which encodes inositol-3-phosphate synthase, cannot be fully repressed by inositol and choline, and UAS(INO1) (inositol-sensitive upstream activating sequence)-driven transcription is enhanced in Acb1p-depleted cells. In addition, the reduction in inositol-mediated repression of INO1 transcription observed after depletion of Acb1p appeared to be independent of the transcriptional repressor, Opi1p. We also demonstrated that INO1 and OPI3 expression can be normalized in Acb1p-depleted cells by the addition of high concentrations of exogenous fatty acids, or by the overexpression of FAS1 or ACC1. Together, these findings revealed an Acb1p-dependent connection between fatty acid metabolism and transcriptional regulation of phospholipid biosynthesis in yeast. Finally, expression of an Acb1p mutant which is unable to bind acyl-CoA esters could not normalize the transcriptional changes caused by Acb1p depletion. This strongly implied that gene expression is modulated either by the Acb1p-acyl-CoA ester complex directly or by its ability to donate acyl-CoA esters to utilizing systems.

Current evidence supporting a role of cannabinoid CB1 receptor (CB1R) antagonists as potential pharmacotherapies for drug abuse disorders.

Since the discovery of the cannabinoid CB1 receptor (CB1R) in 1988, and subsequently of the CB2 receptor (CB2R) in 1993, there has been an exponential growth of research investigating the functions of the endocannabinoid system. The roles of CB1Rs have been of particular interest to behavioral pharmacologists because of their selective presence within the central nervous system (CNS) and because of their association with brain-reward circuits involving mesocorticolimbic dopamine systems. One potential role that has become of considerable recent focus is the ability of CB1Rs to modulate the effects of drugs of abuse. Many drugs of abuse elevate dopamine levels, and the ability of CB1R antagonists or inverse agonists to attenuate these elevations has suggested their potential application as pharmacotherapies for treating drug abuse disorders. With the identification of the selective CB1R antagonist, SR141716, in 1994, and its subsequent widespread availability, there has been a rapid expansion of research investigating its ability to modulate the effects of drugs of abuse. The preliminary clinical reports of its success in retarding relapse in tobacco users have accelerated this expansion. This report critically reviews preclinical and clinical studies involving the ability of CB1R antagonists to attenuate the effects of drugs of abuse, while providing an overview of the neuroanatomical and neurochemical points of contact between the endocannabinoid system and systems mediating abuse-related effects.

Delta9-tetrahydrocannbinol accounts for the antinociceptive, hypothermic, and cataleptic effects of marijuana in mice.

Although it is widely accepted that delta9-tetrahydrocannabinol (delta9-THC) is the primary psychoactive constituent of marijuana, questions persist as to whether other components contribute to marijuana's pharmacological activity. The present experiments assessed the cannabinoid activity of marijuana smoke exposure in mice and tested the hypothesis that delta9-THC mediates these effects through a CB1 receptor mechanism of action. First, the effects of delta9-THC on analgesia, hypothermia, and catalepsy were compared with those of a marijuana extract with equated delta9-THC content after either i.v. administration or inhalation exposure. Second, mice were exposed to smoke of an ethanol-extracted placebo plant material or low-grade marijuana (with minimal delta9-THC but similar levels of other cannabinoids) that were impregnated with varying quantities of delta9-THC. To assess doses, delta9-THC levels in the blood and brains of drug-exposed mice were determined following both i.v. and inhalation routes of administration. Both marijuana and delta9-THC produced comparable levels of antinociception, hypothermia, and catalepsy regardless of the route of administration, and these effects were blocked by pretreatment with the CB1 antagonist SR141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl]. Importantly, the blood and brain levels of delta9-THC were similar in mice exhibiting similar pharmacological effects, regardless of the presence of non-delta9-THC marijuana constituents. The present experiments provide evidence that the acute cannabinoid effects of marijuana smoke exposure on analgesia, hypothermia, and catalepsy in mice result from delta9-THC content acting at CB1 receptors and that the non-delta9-THC constituents of marijuana (at concentrations relevant to those typically consumed) influence these effects only minimally, if at all.

Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.

Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.

Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley rats.

Bisphenol A (BPA) was evaluated at concentrations of 0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm ( approximately 0.001, 0.02, 0.3, 5, 50, and 500 mg/kg/day of BPA) administered in the diet ad libitum to 30 CD((R)) Sprague-Dawley rats/sex/dose for 3 offspring generations, 1 litter/generation, through F3 adults. Adult systemic toxicity at 750 and 7500 ppm in all generations included: reduced body weights and body weight gains, reduced absolute and increased relative weanling and adult organ weights (liver, kidneys, adrenals, spleen, pituitary, and brain), and female slight/mild renal and hepatic pathology at 7500 ppm. Reproductive organ histopathology and function were unaffected. Ovarian weights as well as total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD). Mating, fertility, gestational indices; ovarian primordial follicle counts; estrous cyclicity; precoital interval; gestational length; offspring sex ratios; postnatal survival; nipple/areolae retention in preweanling males; epididymal sperm number, motility, morphology; daily sperm production (DSP), and efficiency of DSP were all unaffected. At 7500 ppm, vaginal patency (VP) and preputial separation (PPS) were delayed in F1, F2, and F3 offspring, associated with reduced body weights. Anogenital distance (AGD) on PND 0 was unaffected for F2 and F3 males and F3 females (F2 female AGD was increased at some doses, not at 7500 ppm, and was considered not biologically or toxicologically relevant). Adult systemic no observed adverse effect level (NOAEL) = 75 ppm (5 mg/kg/day); reproductive and postnatal NOAELs = 750 ppm (50 mg/kg/day). There were no treatment-related effects in the low-dose region (0.001-5 mg/kg/day) on any parameters and no evidence of nonmonotonic dose-response curves across generations for either sex. BPA should not be considered a selective reproductive toxicant, based on the results of this study.

Quantitative analysis of the principle soy isoflavones genistein, daidzein and glycitein, and their primary conjugated metabolites in human plasma and urine using reversed-phase high-performance liquid chromatography with ultraviolet detection.

Soy isoflavones are becoming of increasing interest as nutritional agents which can be used to combat osteoporosis and hyperlipidemia, and are also being considered as potential cancer chemopreventive compounds. However, prior to their formulation and distribution as therapeutic agents, thorough pharmacokinetic and toxicological assessment needs to be completed in men and women in a variety of health conditions in order to ensure their therapeutic efficacy and safety. At this time, studies of purified soy isoflavones are possible, and are being designed to fully evaluate the pharmacological utility of these preparations. In support of these studies, quantitative analysis of soy isoflavones in biological fluids can be accomplished with a wide variety of methods and analytical instrumentation. However, the relatively ubiquitous presence of high-performance liquid chromatography with ultraviolet detection (HPLC-UV) in most analytical laboratories, the relative ease of its operation, and the lesser expense of this instrumentation as compared to more sophisticated techniques such as liquid chromatography-mass spectrometry, offers some distinct advantages for its use in pharmacokinetic studies. In this manuscript, the development and validation of an HPLC-UV method for the quantitation of the principal soy isoflavones, genistein, daidzein, and glycitein, and their primary metabolites, in human plasma and urine is described. This analytical approach allows for pharmacologically relevant concentrations of the analytes and their principle metabolites to be detected, and has been validated in close agreement with the US Food and Drug Administration's guidelines for the validation of methods to be used in support of pharmacokinetic studies.

Disposition of propargyl alcohol in rat and mouse after intravenous, oral, dermal and inhalation exposure.

1. The disposition of propargyl alcohol (PAL) radiolabelled with carbon-14 ([2,3-14C]PAL) was determined in the F344 rat and B6C3F1 mouse following intravenous (i.v.), oral, inhalation and dermal exposure. 2. By 72h following an i.v. (1 mg kg(-1) or oral (50 mg kg(-1) dose, 76-90% of the dose was excreted. Major routes of excretion by rat were urine (50-62%), CO2 (19-26%) and faeces (6-14%). Major routes of exerection by mouse were urine (30-40%), CO2 (22-26%) and faeces (10-20%). Less than 6% of the dose remained in tissues at 72 h. Biliary exeretion of radioactity by rat (62% in 4 h) was much greater than elimination in faeces (6% in 72 h), indicating that PAL metabolites underwent extensive enterohepatic recycling. 3. Dermal exposure studies demonstrated that dermal absorption of PAL was minimal due to its inherent volatility. 4. In the inhalation studies (1, 10 or 100 ppm for 6 h), 23-68% of the radioactivity to which animals were exposed was absorbed. The primary route of excretion was urine (23-53%), and significant portion was exhaled as volatile organics (15-30%). 5. PAL was extensively metabolized by both species. One metabolite was identified as 3,3-bis[(2-(acetylamino)-2-carboxyethyl)thio]-1-propanol, which is consistent with Banijamali et al. (1999).

Metabolism and disposition of alpha-methylstyrene in rats.

alpha-Methylstyrene (AMS) is a volatile hydrocarbon used primarily in the production of specialty polymers and resins. In the present study, the tissue distribution, metabolism, and excretion of [(14)C]AMS was investigated in male rats after i.v. administration (11 mg/kg). Over 90% of AMS administered intravenously to rats was excreted in 72 h. Urinary excretion accounted for 86% of the administered dose, volatile breath and feces accounted for 2.2 and 1.9%, respectively, and elimination as carbon dioxide was negligible. Metabolites were isolated from rat urine following a high oral dose of AMS (1000 mg/kg) and characterized using gas chromatography/mass spectrometry and NMR spectrometry. The metabolites were 2-phenyl-1,2-propanediol (3% of urinary radioactivity) and its glucuronide (50%), atrolactic acid (27%), S-(2-hydroxy-2-phenylpropyl)-N-acetylcysteine (13%), and 2-phenylpropionic acid (1%); the glucuronides and mercapturates were each conjugated on the methylene carbon beta to the ring. The presence of both of the diastereomeric isomers of the mercapturates and of the glucuronides suggested that the initial epoxidation of AMS was not stereoselective and proceeded with addition of active oxygen to yield enantiomeric epoxides. Incubation of AMS with human liver slices produced the same metabolites as those excreted in rat urine, with 2-phenyl-1,2-propanediol present as the predominant metabolite after 5 h of incubation.

QSAR analysis of Delta(8)-THC analogues: relationship of side-chain conformation to cannabinoid receptor affinity and pharmacological potency.

A novel quantitative structure-activity relationship (QSAR) for the side-chain region of Delta(8)-tetrahydrocannabinol (Delta(8)-THC) analogues is reported. A series of 36 side-chain-substituted Delta(8)-THCs with a wide range of pharmacological potency and CB1 receptor affinity was investigated using computational molecular modeling and QSAR analyses. The conformational mobility of each compound's side chain was characterized using a quenched molecular dynamics approach. The QSAR techniques included a modified active analogue approach (MAA), multiple linear regression analyses (MLR), and comparative molecular field analysis (CoMFA) studies. All three approaches yielded consistent results. The MAA approach applied to a set of alkene/alkyne pairs identified the most active conformers as those with conformational mobility constrained within an approximately 8 A radius. MLR analyses (restricted to 15 hydrocarbon side-chain analogues) identified two variables describing side-chain length and terminus position that were able to fit the pharmacological data for receptor affinity with a correlation coefficient for pK(D) of 0.82. While chain length was found to be directly related to receptor affinity, the angle made by the side chain from its attachment point to its terminus (angle defined by C3-C1'-side-chain terminus carbon, see Figure 1) was found to be inversely related to affinity. These results suggest that increased side-chain length and increased side-chain ability to wrap around the ring system are predicted to increase affinity. Therefore, the side chain's conformational mobility must not restrict the chain straight away from the ring system but must allow the chain to wrap back around toward the ring system. Finally, the CoMFA analyses involved all 36 analogues; they also provided data to support the hypothesis that for optimum affinity and potency the side chain must have conformational freedom that allows its terminus to fold back and come into proximity with the phenolic ring.

Effects of sex and gonadectomy on cocaine metabolism in the rat.

The purpose of the current study is to determine whether sex differences in metabolism of cocaine (COC) exist that could contribute to the greater behavioral sensitivity of females to COC administration. To investigate this question, concentrations of COC and its two principle metabolites benzoylecgonine (BE) and ecgonine methyl ester (EME) were measured by gas chromatography/mass spectroscopy in brain and plasma collected from male and female rats that were sacrificed between 5 and 90 min after injection COC (15 mg/kg i.p.). COC concentrations did not differ in plasma or brain tissue of males and females, but sex-specific patterns of metabolite distribution were detected. BE was 2-fold higher in plasma and brain of males than females, whereas EME was much higher in brain and plasma of females. The influence of gonadal hormones on COC metabolite patterns were determined using gonadectomized and prepubertal rats. Castration of male or female rats did not alter brain or plasma COC, but did decrease BE concentrations. Seven-day-old pups injected with 15 mg/kg of COC had higher blood and brain COC than adults and relatively low levels of metabolites. No sex differences were found for COC, BE, or EME in brain or plasma of pups. These findings indicate that although gonadal steroids influence COC metabolism, these effects do not explain sex differences in COC-induced behaviors.

Identification of urinary metabolites of isoprene in rats and comparison with mouse urinary metabolites.

Isoprene, a major commodity chemical used in production of polyisoprene elastomers, has been shown to be carcinogenic in rodents. Similar to findings for the structurally related compound butadiene, mice are more susceptible than rats to isoprene-induced toxicity and carcinogenicity. Although differences in uptake, and disposition of isoprene in rats and mice have been described, its in vivo biotransformation products have not been characterized in either species. The purpose of these studies was to identify the urinary metabolites of isoprene in Fischer 344 rats and compare these metabolites with those formed in male B6C3F1 mice. After i.p. administration of 64 mg [14C]isoprene/kg to rats and mice, isoprene was excreted unchanged in breath ( approximately 50%) or as urinary metabolites ( approximately 32%). In rats isoprene was primarily excreted in urine as 2-hydroxy-2-methyl-3-butenoic acid (53%), 2-methyl-3-buten-1,2-diol (23%), and the C-1 glucuronide conjugate of 2-methyl-3-buten-1,2-diol (13%). These metabolites are consistent with preferential oxidation of isoprene's methyl-substituted vinyl group. No oxidation of the unsubstituted vinyl group was observed. In addition to the isoprene metabolites found in rat urine, mouse urine contained numerous other isoprene metabolites with a larger percentage (25%) of total urinary radioactivity associated with an unidentified, polar fraction than in the rat (7%). Unlike butadiene, there was no evidence that glutathione conjugation played a significant role in the metabolism of isoprene in rats. Because of the unidentified metabolites in mouse urine, involvement of glutathione in the metabolism of isoprene in mice cannot be delineated.

Isolation and identification of novel metabolites of gemfibrozil in rat urine.

Gemfibrozil (GEM) is a clofibrate analog used to treat moderate to severe hypertriglyceridemias. In lab animals, GEM causes peroxisome proliferation, an effect that has been associated with hepatocarcinogenesis in rats. In humans, hepatobiliary disorders, but not carcinogenesis, have been associated with GEM therapy. In the present study [14C]GEM was administered orally to rats at a dose of 2000 mg/kg. At various time points, radioactivity in urine was analyzed by liquid scintillation spectrometry, high-pressure liquid chromatography, liquid chromatography/mass spectrometryn, gas chromatography/mass spectroscopy, and nuclear magnetic resonance. Nine metabolites of GEM were identified, some that have not been reported previously. Although the majority of metabolites were glucuronidated, some nonglucuronidated metabolites were identified in urine, including a diol metabolite (both ring methyls hydroxylated), and the product of its further metabolism, the acid-alcohol derivative (ortho ring methyl hydroxylated, meta ring methyl completely oxidized to the acid). Hydroxylation of the aromatic ring also was a common pathway for GEM metabolism, leading to the production of two phenolic metabolites, only one of which was detected in the urine in the nonconjugated or free form. Also of interest was the finding that both acyl and ether glucuronides were produced, including both glucuronide forms of the same metabolite (e. g., 1-O-GlcUA, 5'-COOH-GEM, and 5'-COO-GlcUA-GEM); the positions and functionality of the glucuronide conjugates were identified using base hydrolysis or glucuronidase treatment, in combination with liquid chromatography/MSn and nuclear magnetic resonance.

The bioactive conformation of aminoalkylindoles at the cannabinoid CB1 and CB2 receptors: insights gained from (E)- and (Z)-naphthylidene indenes.

The aminoalkylindoles (AAIs) are agonists at both the cannabinoid CB1 and CB2 receptors. To determine whether the s-trans or s-cis form of AAIs is their receptor-appropriate conformation, two pairs of rigid AAI analogues were studied. These rigid analogues are naphthylidene-substituted aminoalkylindenes that lack the carbonyl oxygen of the AAIs. Two pairs of (E)- and (Z)-naphthylidene indenes (C-2 H and C-2 Me) were considered. In each pair, the E geometric isomer is intended to mimic the s-trans form of the AAIs, while the Z geometric isomer is intended to mimic the s-cis form. Complete conformational analyses of two AAIs, pravadoline (2) and WIN-55, 212-2 (1), and of each indene were performed using the semiempirical method AM1. S-trans and s-cis conformations of 1 and 2 were identified. AM1 single-point energy calculations revealed that when 1 and each indene were overlayed at their corresponding indole/indene rings, the (E)- and (Z)-indenes were able to overlay naphthyl rings with the corresponding s-trans or s-cis conformer of 1 with an energy expense of 1.13/0.69 kcal/mol for the C-2 H (E/Z)-indenes and 0.82/0.74 kcal/mol for the C-2 Me (E/Z)-indenes. On the basis of the hypothesis that aromatic stacking is the predominant interaction of AAIs such as 1 at the CB receptors and on the demonstration that the C-2 H (E/Z)- and C-2 Me (E/Z)-indene isomers can mimic the positions of the aromatic systems in the s-trans and s-cis conformers of 1, the modeling results support the previously established use of indenes as rigid analogues of the AAIs. A synthesis of the naphthylidene indenes was developed using Horner-Wittig chemistry that afforded the Z isomer in the C-2 H series, which was not produced in significant amounts from an earlier reported indene/aldehyde condensation reaction. This approach was extended to the C-2 Me series as well. Photochemical interconversions in both the C-2 H and C-2 Me series were also successful in obtaining the less favored isomer. Thus, the photochemical process can be used to provide quantities of the minor isomers C-2 H/Z and C-2 Me/E. The CB1 and CB2 affinities as well as the activity of each compound in the twitch response of the guinea pig ileum (GPI) assay were assessed. The E isomer in each series was found to have the higher affinity for both the CB1 and CB2 receptors. In the rat brain membrane assay versus [3H]CP-55,940, the Ki's for the C-2 H/C-2 Me series were 2.72/2.89 nM (E isomer) and 148/1945 nM (Z isomer). In membrane assays versus [3H]SR141716A, a two-site model was indicated for the C-2 H/C-2 Me (E isomers) with Ki's of 10. 8/9.44 nM for the higher-affinity site and 611/602 nM for the lower-affinity site. For the Z isomers, a one-site model was indicated with Ki's of 928/2178 nM obtained for the C2 H/C-2 Me analogues, respectively. For the C-2 H/C-2 Me series, the CB2 Ki's obtained using a cloned cell line were 2.72/2.05 nM (E isomer) and 132/658 nM (Z isomer). In the GPI assay, the relative order of potency was C-2 H E > C-2 Me E > C-2 H Z > C-2 Me Z. The C-2 H E isomer was found to be equipotent with 1, while the C-2 Me Z isomer was inactive at concentrations up to 3.16 microM. Thus, results indicate that the E geometric isomer in each pair of analogues is the isomer with the higher CB1 and CB2 affinities and the higher pharmacological potency. Taken together, results reported here support the hypothesis that the s-trans conformation of AAIs such as 1 is the preferred conformation for interaction at both the CB1 and CB2 receptors and that aromatic stacking may be an important interaction for AAIs at these receptors.

Comparative receptor binding analyses of cannabinoid agonists and antagonists.

To further characterize neuronal cannabinoid receptors, we compared the ability of known and novel cannabinoid analogs to compete for receptor sites labeled with either [3H]SR141716A or [3H]CP-55,940. These efforts were also directed toward extending the structure-activity relationships for cannabinoid agonists and antagonists. A series of alternatively halogenated analogs of SR141716A were synthesized and tested in rat brain membrane binding assays along with the classical cannabinoids, Delta9-tetrahydrocannabinol, cannabinol, cannabidiol, the nonclassical cannabinoid CP-55,940, the aminoalkylindole WIN55212-2 and the endogenous fatty acid ethanolamide, anandamide. Saturation binding isotherms were performed with both radioligands, as were displacement studies, allowing an accurate comparison to be made between the binding of these various compounds. Competition studies demonstrated that all of the compounds were able to displace the binding of [3H]CP-55,940 with rank order potencies that agreed with previous studies. However, the rank order potencies of these compounds in competition studies with [3H]SR141716A differed significantly from those determined with [3H]CP-55,940. These results suggest that CP-55,940, WIN55212-2 and other agonists interact with cannabinoid binding sites within the brain which are distinguishable from the population of binding sites for SR141716A, its analogs and cannabidiol. Structural modification of SR141716A significantly altered the affinity of the compound and its relative ability to displace either [3H]CP-55,940 or [3H]SR141716A preferentially within the rat brain receptor membrane preparation.

Synthesis and pharmacological comparison of dimethylheptyl and pentyl analogs of anandamide.

(Dimethylheptyl)anandamide [(16,16-dimethyldocosa-cis-5,8,11,14-tetraenoyl)ethanolamine ] (17a) and its amide analogs were synthesized by Wittig coupling of a ylide derived from a fragment of arachidonic acid. These amides were compared to the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide, 2a) and its amide analogs in pharmacological assays for potential enhancement of cannabimimetic activities. The receptor affinity to rat brain membranes of the dimethylheptyl (DMH) analogs increased by an order of magnitude in most comparisons to the corresponding anandamides in displacement assays versus the cannabinoid agonist [3H]CP 55,940 or antagonist [3H]SR141716A, for which rank order differences in affinity were observed. An order of magnitude enhancement of potency with comparable or higher efficacy in behavioral assays in the mouse tetrad of tests of cannabinoid activity was observed in 17a versus 2a. In contrast, no enhancement in potency for the pentyl to DMH side chain exchange was seen in the mouse vas deferens assay. The data indicate a structural equivalence between classical plant cannabinoids and 2a as well as different receptor-ligand interactions that characterize multiple receptor sites or binding modes.

Synthesis, pharmacology, and molecular modeling of novel 4-alkyloxy indole derivatives related to cannabimimetic aminoalkyl indoles (AAIs).

Several novel 4-alkyloxy-aminoalkyl indole derivatives 3 were synthesized from 4-benzyloxyindole (1). Alkylation of 1 with 4-(2-chloroethyl)morpholine (NaH/HMPA) formed 2. Deprotection using palladium hydroxide on carbon/hydrogen followed by alkylation with the appropriate alkyl bromide gave the target compounds 3b-3j. In the synthesis of 3i and 3j, the appropriate alkyl bromides 13 and 17 were prepared from the commercially available 1-naphthylethyl bromide 9 using the chain lengthening sequences as shown in Scheme 3. In receptor binding assay and in vivo testing, the long chain alkoxy compounds 3g and 3h (Ki = 127 nM) showed affinity for the CB1 receptor which was approximately 16-35-fold less than that of WIN 55,225. However, the pharmacological profile of 3h mimics that of WIN 55,212. An examination of the SAR of these analogues shows that translocating the napthyl group in AAIs from the C-3 position to C-4 via an oxygen (ether linkage) decreases activity which is in contrast to previous findings that a naphthylcarbonyl at C-4 retains activity. The present work points to the importance of the role of a keto group in the interaction with the receptor. Molecular modeling work suggests that, although reasonable superposition of key structural features between delta 9-THC and AAIs can be made, the overlay is not straightforward. The present study also illustrates the difficulty in accommodating AAIs into the cannabinoid pharmacophore and it seems likely that a unique pharmacophore will need to be developed. Only then will the similarities to and differences from the classical cannabinoid pharmacophore be clearly delineated.

Lauramide diethanolamine absorption, metabolism, and disposition in rats and mice after oral, intravenous, and dermal administration.

The disposition of carbon-14-labeled lauramide diethanolamine (LDEA) was determined in rats after iv, dermal, and oral administration, and in mice after iv and dermal administration. Intravenous doses of LDEA to rats and mice (25 and 50 mg/kg, respectively) were mostly excreted in the urine (ca. 80-90%), with only about 10% excreted in the feces 72 hr after dosing. No unchanged LDEA, diethanolamine, or diethanolamine-derived metabolites were detected in urine. LDEA concentrated to the highest levels in the adipose tissue, and was only very slowly cleared from that tissue. Residues were also observed in liver and kidney, but clearance from those tissues paralleled the decreases in blood concentrations. Incubations of LDEA with liver slices from rats and humans showed that the compound is well absorbed by hepatic tissue from both species. LDEA was readily converted to metabolites found in vivo in rats, as well as other metabolites that are potentially intermediate products formed after omega- and/or omega-1 to 4 hydroxylation. Treatment with diethylhexylphthalate, an inducer of cytochrome P4504A1, which catalyzes the omega-hydroxylation of lauric and other fatty acids, demonstrated the involvement of that isozyme in the hydroxylation of LDEA. Dermally applied LDEA, at doses of 25 and 400 mg/kg to rats, was moderately (25-30%) well absorbed. Repeat administration (25 mg/kg/day for 3 weeks) did not change the rate of LDEA absorption. The absorption of 100 mg/kg doses was studied in jugular vein-cannulated rats. Steady state levels of LDEA equivalents were reached 24 hr after dermal administration. LDEA comprised about 15% of the radioactivity in plasma, with the remainder present as polar metabolites. A range of 50-70% of the dermal doses to mice, applied at 50, 100, 200, and 800 mg/kg, was absorbed in 72 hr. Absorbed LDEA distributed into the tissues with the same relative profile as that for the iv dose, except that distribution into adipose tissue was considerably lower. High oral doses of LDEA (100 mg/kg) in rats were well absorbed and mostly excreted in the urine as two very polar metabolites. The metabolites were isolated and characterized as the half-acid amides of succinic and of adipic acid, presumably arising from omega-hydroxylation and eventual beta-oxidation to give the chain-shortened products.