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Biomedical subjects

A Makriyannis

Publications and source records attributed to A Makriyannis.

At least 73 records · Page 4Linked to original sources
PubMed1998

Structural requirements for arachidonylethanolamide interaction with CB1 and CB2 cannabinoid receptors: pharmacology of the carbonyl and ethanolamide groups.

Analogs of arachidonylethanolamide (anandamide) were prepared to investigate the structural requirements for ligand binding to and activation of the CB1 and CB2 cannabinoid receptors. The importance of the presence and the placement of the carbonyl was examined with analogs lacking the carbonyl or with the carbonyl amide order reversed. The presence and location of the carbonyl is essential for high-affinity binding to both cannabinoid receptor subtypes, and for determination of signal transduction via G-proteins. Methyl groups were substituted on the 1'- and 2'-positions of arachidonylethanolamide and the significance of chirality was examined. Stereochemical differences in the ethanolamide group influence the affinity for both cannabinoid receptor subtypes and the signal transduction capabilities of the methanandamide derivatives.

Adenylyl Cyclases↗
PubMed1998

Imaging the brain marijuana receptor: development of a radioligand that binds to cannabinoid CB1 receptors in vivo.

The major active ingredient of marijuana, (-)-delta9-tetrahydrocannabinol, exerts its psychoactive effects via binding to cannabinoid CB1 receptors, which are widely distributed in the brain. Radionuclide imaging of CB1 receptors in living human subjects would help explore the presently unknown physiological roles of this receptor system, as well as the neurochemical consequences of marijuana dependence. Currently available cannabinoid receptor radioligands are exceedingly lipophilic and unsuitable for in vivo use. We report the development of a novel radioligand, [123I]AM281[N-(morpholin-4-yl)-5-(4-[123I]iodophenyl)-1-(2,4 -dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], that is structurally related to the CB1-selective antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide]. Baboon single photon emission computed tomography studies, mouse brain dissection studies, and ex vivo autoradiography in rat brain demonstrated rapid passage of [123I]AM281 into the brain after intravenous injection, appropriate regional brain specificity of binding, and reduction of binding after treatment with SR141716A. AM281 has an affinity in the low nanomolar range for cerebellar binding sites labeled with [3H]SR141716A in vitro, and binding of [123I]AM281 is inhibited by several structurally distinct cannabinoid receptor ligands. We conclude that [123I]AM281 has appropriate properties for in vivo studies of cannabinoid CB1 receptors and is suitable for imaging these receptors in the living human brain.

Animals↗
PubMed1998

Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors.

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R.

Animals↗
PubMed1998

Effects of delta-9-tetrahydrocannabinol and (R)-methanandamide on open-field behavior in rats.

This study compared the effects of (R)-methanandamide, an analog of the mammalian brain constituent anandamide, and delta-9-tetrahydrocannabinol on the open-field behavior of male Sprague-Dawley rats. Rats were individually housed with free access to food and water. Animals were treated with 0, 1, 3, and 5.6 mg/kg delta-9-tetrahydrocannabinol given i.p. 30 min pre-session; and 0, 3, 10, and 18 mg/kg (R)-methanandamide, 15 min pre-session. The behavioral categories recorded were ambulation (the number of squares crossed), rearing (the number of times the rat stood erect on its hind-legs), latency (the time in seconds to leave the starting area, the circle in the center of the field), circling (the number of times the animal turned around its vertical axis, 0.5 point given for each 180 degrees turn), grooming (the number of cleaning bouts), urination and defecation (the number of urination spots and fecal boli deposited during the 5 min observation period). Delta-9-tetrahydrocannabinol was more potent than (R)-methanandamide, but otherwise the effects of delta-9-tetrahydrocannabinol and (R)-methanandamide were similar, with one exception; whereas delta-9-tetrahydrocannabinol produced dose-related increases in circling, (R)-methanandamide did not increase circling over the doses examined. The delta-9-tetrahydrocannabinol-induced increase in circling was blocked by the central cannabinoid receptor CB1 antagonist SR 141716. The differential effects with regard to circling may indicate that there are qualitative behavioral differences in the effects of delta-9-tetrahydrocannabinol and (R)-methanandamide.

Analysis of Variance↗
PubMed1997

Inhibition of intestinal motility by anandamide, an endogenous cannabinoid.

The endogenous cannabinoid ligand anandamide (arachidonylethanolamide) inhibited the intestinal passage of a charcoal meal when administered s.c. in mice at doses ranging from 0.1 to 50 mg/kg. This effect was prevented by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] (1 mg/kg s.c.), but it was not affected by the anandamide transport inhibitor, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (50 mg/kg, s.c.). The results indicate that anandamide modulates intestinal motility in mice by activating cannabinoid CB1 receptors. They also suggest that anandamide transport, which was previously shown to participate in terminating neural and vascular responses to anandamide, does not contribute to anandamide inactivation in intestinal tissue.

Animals↗
PubMed1997

Effect of the cannabinoid receptor SPECT agent, AM 281, on hippocampal acetylcholine release from rat brain slices.

The SPECT ligand AM 281, a less lipophilic analog of the cannabinoid receptor antagonist SR 141716A, robustly potentiated electrically-evoked release of acetylcholine from superfused hippocampal slices and prevented the inhibition of acetylcholine release by the cannabimimetic drug WIN 55212-2. These results, similar to earlier observations with SR 141716A, indicate that AM 281 is either a cannabinoid receptor antagonist or inverse agonist. Despite showing lower affinity than SR 141716A in hippocampal membrane binding experiments, AM 281 had slightly greater potency than SR 141716A in the hippocampal slice experiments, perhaps because of reduced drug absorption to slice membranes and to the apparatus.

Acetylcholine↗
PubMed1997

Potentiation of anandamide hypotension by the transport inhibitor, AM404.

The putative endogenous cannabinoid, anandamide (0.2-2 mg/kg i.v.), decreased systemic blood pressure dose-dependently in anesthesized guinea pigs. These effects were prevented by the CB1 cannabinoid receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] at the dose of 0.2 mg/kg i.v. The vasodepressor responses to anandamide were significantly potentiated and prolonged by a novel inhibitor of carrier-mediated anandamide transport, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (10 mg/kg, i.v.). These results suggest that anandamide transport participates in terminating the vascular actions of anandamide.

Animals↗
PubMed1997

Functional role of high-affinity anandamide transport, as revealed by selective inhibition.

Anandamide, an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic hydrolysis. The compound N-(4-hydroxyphenyl)arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes in vitro, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid receptors or inhibit anandamide hydrolysis, it enhanced receptor-mediated anandamide responses in vitro and in vivo. The data indicate that carrier-mediated transport may be essential for termination of the biological effects of anandamide, and may represent a potential drug target.

Analgesics↗
PubMed1997

Time course of the effects of different cannabimimetics on prolactin and gonadotrophin secretion: evidence for the presence of CB1 receptors in hypothalamic structures and their involvement in the effects of cannabimimetics.

Several reports have demonstrated that (-)-delta9-tetrahydrocannabinol (delta9-THC) and arachidonylethanolamide [anandamide (AEA)] were able to inhibit prolactin (PRL) secretion from the anterior pituitary gland in male rodents, whereas ovarian phase-dependent effects were seen in females. However, in most of these studies, the analysis of PRL levels was performed at times longer than 30 min after cannabinoid administration. In the present study, we examined the time course of the effects of three different cannabimimetics, delta9-THC, AEA, and AM356 (R-methanandamide), a more stable analog of AEA, on PRL and gonadotrophin secretion in male Wistar rats. In addition, we characterized the presence of cannabinoid receptors in hypothalamic structures related to neuroendocrine control and studied their potential involvement in the effects of cannabimimetics. We found that the three compounds decreased plasma luteinizing hormone (LH) levels, although only the effects of delta9-THC were statistically significant. The inhibitory effect was already apparent at 40 min after administration, but only in the case of delta9-THC did it persist up to 180 min after administration. No significant changes were seen in plasma follicle-stimulating hormone (FSH) levels after the administration of any of the three different cannabimimetics at any of the four times analyzed. Both AEA and AM356 produced a significant decrease in plasma PRL levels, which appeared at 20 min after administration and persisted up to 60 min, waning after this time. Interestingly, the time course of the effect of delta9-THC resembled that of AEA and AM356 only during the later part of the response, because delta9-THC produced a marked increase in plasma PRL levels at 20 min, no changes at 40 min and a decrease from 60 min up to 180 min. In additional experiments, we tried to elucidate which of these two phases observed after delta9-THC administration was mediated by the activation of cannabinoid receptors. These receptors are present in hypothalamic structures related to neuroendocrine control, with the highest densities in the arcuate nucleus (dorsal area) and the medial preoptic area, and the lowest in the lateral hypothalamic area, although none of these regions exhibited high densities for this receptor as compared with classical regions containing cannabinoid receptors, such as the basal ganglia. The activation of these receptors by delta9-THC seems to be involved in the inhibitory phase of the effect of this cannabinoid on PRL release, but not in the early stimulation; when these receptors were blocked with a specific antagonist, SR141716, the stimulation by delta9-THC was still observed, but the late inhibition was abolished. In summary, AEA and AM356 markedly decreased PRL release and slightly decreased LH secretion, with no changes on FSH release. delta9-THC also produced a marked inhibition of LH secretion, but its effects on PRL were biphasic with an early stimulation not mediated by the activation of cannabinoid receptors, followed by a late and cannabinoid receptor-mediated inhibition. Their site of action may well be the hypothalamic structures related to neuroendocrine control, which contain a small, but probably very active, population of cannabinoid receptors.

Animals↗
PubMed1997

Syntheses of 1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC) and analogs with 13C- and 2H-labeled choline head groups.

The syntheses of four head group labeled analogs of 1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC) (6) by a general method from 1,2-di-O-palmitoyl-sn-glycero-3-phosphatidic acid (5) have been performed. The syntheses of 1,2-di-O-palmitoyl-sn-glycero-3-phospho[alpha-13C]choline (6a) and 1,2-di-O-palmitoyl-sn-glycero-3-phospho[beta-13C]choline (6b) were performed from labeled [1-13C]glycine (1a) in 52% overall yield and from [2-13C]glycine (1b) in 56% overall yield, respectively. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[N(C2H3)3]choline (9) was prepared from 2-aminoethanol in 39% overall yield. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[alpha-C2H2]choline (12) was prepared from N,N-dimethylglycine ethyl ester in 50% overall yield.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

AM630 antagonism of cannabinoid-stimulated [35S]GTP gamma S binding in the mouse brain.

This research was designed to determine the action of the novel aminoalkylindole AM630 (6-iodo-pravadoline) at the cannabinoid receptor by studying its interaction with the cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-y]-(1-naphthalenyl)methanone mesylate) on guanosine-5'-O-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding in mouse brain. WIN 55,212-2 stimulated [35S]GTP gamma S binding, while AM630 had no effect. AM630 antagonized WIN 55,212-2-2induced [35S]GTP gamma S binding and shifted the WIN 55,212-dose-response curve to the right. These results clearly demonstrate that AM630 exerts cannabinoid receptor antagonist properties in the brain.

Analgesics↗
PubMed1997

Fatty acid sulfonyl fluorides inhibit anandamide metabolism and bind to the cannabinoid receptor.

Arachidonoyl ethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors (CB1, CB2) and a putative neurotransmitter. Phenylmethylsulfonyl fluoride (PMSF) is an inhibitor of the enzyme (an amidase) which hydrolyzes anandamide to arachidonic acid and ethanolamine. We report here that fatty acid sulfonyl fluorides are potent inhibitors of anandamide metabolism. In order to investigate the SAR of these anandamide amidase inhibitors we tested a series of fatty acid (C12 to C20) sulfonyl fluorides both as inhibitors of anandamide degradation and as ligands for the central cannabinoid receptor (CB1). AM374 (palmitylsulfonyl fluoride, C16) was approximately 20 times more potent than PMSF and 50 times more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain homogenates. AM374 was over a thousand-fold more effective than PMSF in inhibiting the amidase in cultured cells. The C12 to C18 sulfonyl fluoride analogs were equipotent as inhibitors of the amidase and the reverse reaction (the synthase) with nanomolar IC50 values. These compounds generally showed decreasing affinity for the CB1 receptor as the chain length increased; thus, C12 sulfonylfluoride had an IC50 of 18 nM and C20 sulfonylfluoride had an IC50 of 78 microM. The C14, C16, and C18 sulfonyl fluorides showed high selectivity for the amidase over the CB1 receptor and thus are potentially useful selective anandamide amidase inhibitors.

Amidohydrolases↗
PubMed1997

Differential scanning calorimetry and small angle x-ray diffraction study of the interaction of (R)-PAF, (R)-ET-18-OME and (R)-Lyso-PAF with model membranes.

(R)-PAF, (R)-ET-18-OMe and (R)-Lyso-PAF are ether phospholipid analogs exhibiting different biological activities. All three molecules are highly lipophilic and interact with cell membranes. The manner in which their interactions occur is relevant to their biological activities and may provide information on their mechanism(s) of action at the molecular level. We have studied the interactions of (R)-PAF, (R)-ET-18-OMe and (R)-Lyso-PAF with model membranes using differential scanning calorimetry (DSC). A concentration-dependence study with DSC shows that all three analogs induce similar changes in the thermotropic behavior of model membranes. All produce a slight decrease in the main phase transition temperature of DPPC bilayers (deltaTc = 0.3 to 1.4 degrees C) but have a more substantial effect on the pretransition (deltaTc = 0.8 to 4.5 degrees C). All three disorder phosphatidylcholine (PC) membranes but impart order to PC/cholesterol membranes. We have used small angle x-ray diffraction experiments to supplement the DSC evidence on the topography of the ether lipid analogs in the bilayers. All three appear to insert themselves in the bilayer with their long chain parallel with the membrane bilayer chains.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

AM630 is a competitive cannabinoid receptor antagonist in the guinea pig brain.

AM630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [35S]GTPgammaS binding suggesting that difference in AM630 activity in different tissues is not due to species variation.

Analgesics↗
PubMed1997

Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors.

The binding of [123I]AM251 (a radioiodinated analog of the cannabinoid CB1 receptor antagonist SR141716A) was compared to that of [3H]CP 55,940 in mouse and rat brain preparations. Scatchard analysis of the binding of [123I]AM251 and [3H]CP 55,940 to membranes prepared from mouse cerebellum, striatum and hippocampus yielded similar Bmax values (15-41 pmol/g wet wt tissue). Kd values were lower for [123I]AM251 (0.23-0.62 nM) than for [3H]CP 55,940 (1.3-4 nM). CP 55,940 and SR141716A increased dissociation of [123I]AM251 from binding sites in mouse cerebellar homogenates to a similar extent. The structurally dissimilar cannabinoid receptor ligands THC, methanandamide, WIN 55, 212-2, CP 55,940 and SR141716A were each able to fully compete with binding of both [123I]AM251 and [3H]CP 55,940 in mouse cerebellum. In vitro autoradiography demonstrated that the distribution of binding sites for [123I]AM251 in rat brain was very similar to published distributions of binding sites for [3H]CP 55,940. Together, these observations suggest that AM251 binds to the same site (the cannabinoid CB1 receptor) in rodent brains as CP 55,940. However, the binding site domains which interact with AM251 and CP 55,940 may not be identical, since IC50 values for cannabinoid receptor ligands depended on whether [123I]AM251 or [3H]CP 55,940 was used as radioligand.

Animals↗
PubMed1996

The conformational properties of the antineoplastic ether lipid 1-thiohexadecyl-2-O-methyl-S-glycero-3-phosphocholine.

Thio analogs of platelet activating factor (PAF) are of great interest because they exhibit antineoplastic properties both in vitro and in vivo. In contrast to most known anticancer agents, these lipids appear not to act through the synthesis and function of DNA and, therefore, offer a new avenue of approaching cancer chemotherapy. We have examined the conformational properties of 1-thiohexadecyl-2-O-methyl-S-glycero-3-phosphocholine (ET-S-16-OCH3) in organic solvents and in micelles. The conformational analysis was based on a combination of 1D, 2D NMR spectroscopy and molecular graphics. 1H and 13C spin lattice relaxation time (T1) experiments were also performed to study the dynamic properties of this molecule. The picture emerging from these studies is as follows. The alkyl chain of ET-S-16-OCH3 is the most mobile part of the molecule both in CDCl3/CD3OD and in micelles and exists as a mixture of interconverting conformers including an extended all trans and several low energy conformers with one or more gauche segments. This creates a twisting of the chain and facilitates a spatial communication between the alkyl chain and the glycerol backbone as well as between the alkyl chain and the headgroup. The methylene groups of the thioglycerol backbone and the headgroup are the least mobile while the methine group of the thioglycerol backbone appears to have an intermediate mobility. The conformation of the thioether lipid in the two media may be of relevance during its interaction with its site of action, the cellular membrane. Such a conformation may also play an important role in determining the selectivity of this interaction with different cell membranes.

Antineoplastic Agents↗
PubMed1996

Head group analogs of arachidonylethanolamide, the endogenous cannabinoid ligand.

Several analogs of an endogenous cannabimimetic, arachidonylethanolamide (anandamide), were synthesized to study the structural requirements of the ethanolamide head group. CB1 receptor affinities of the analogs were evaluated by a standard receptor binding assay using tritiated CP-55,940 as the radioligand and compared to anandamide which was shown to have a Ki of 78 nM. Replacement of the amide carbonyl oxygen by a sulfur atom had a detrimental effect on the CB1 affinity. The thio analogs of both anandamide and (R)-methanandamide showed very weak affinity for CB1. The secondary nature of the amidic nitrogen was also shown to be important for affinity, indicating a possible hydrogen-bonding interaction between the amide NH and the receptor. Introduction of a phenolic moiety in the head group resulted in the loss of receptor affinity except when a methylene spacer was introduced between the amidic nitrogen and the phenol. A select group of analogs were also tested for their affinity for the CB2 receptor using a mouse spleen preparation and were found to possess low affinities for the CB2 sites. Notably, anandamide and (R)-methanandamide demonstrated high selectivity for the CB1 receptor. Overall, the data presented here show that structural requirements of the head group of anandamide are rather stringent.

Adjuvants, Immunologic↗