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We have previously studied and compared the location of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) with that of O-methyl-delta 8-THC (Me delta 8-THC) in the membrane using partially hydrated dimyristoylphosphatidylcholine (DMPC) bilayers ((Mavromoustakos et al. (1990) Biophys. Acta 1024, 336-344; Yang et al. (1993) Life Sci. 53, 117-122). delta 8-THC was found to be located near the membrane interface with its phenolic hydroxyl group anchored near the carbonyl groups of DMPC while the more lipophilic Me-delta 8-THC is located deeper in the membrane bilayer. Parallel experiments using Me-delta 8-THC and its 5'-iodo analog (5'-I-Me-delta 8-THC) allowed us to determine the topography of these two molecules in the bilayer. Our results from small angle X-ray diffraction and differential scanning calorimetry (DSC) combined with previous data on the orientation of Me-delta 8-THC in model membranes, led us to the conclusion that these molecules intercalate between contiguous acyl chains in the lipophilic moiety of the membrane bilayer. The terminal iodo group in 5'-I-Me-delta 8-THC was found to reside in a region extending approx. +/- 5 A from the center of the bilayers. The location of Me-delta 8-THC in the membranes as well as its orientation may explain its inability to effectively perturb the bilayer lipid chains.

Binding and photoactivation of a cannabinoid-derived ligand to intact mouse peritoneal cells has resulted in the labelling of a G-protein gamma-subunit. The assignment of structure is based on the product's physical characteristics and its ability to react with a polyclonal antiserum raised against the partial amino acid sequence for a gamma-subunit expressed in spleen cells. The binding characteristics of the ligand to the cells suggests that this gamma-subunit, and its associated alpha and beta subunits, are located in close proximity to one of the transmembrane cannabinoid receptors. Our findings further suggest a possible experimental approach to identifying receptor-G-protein complexes in intact cells.

In order to explore the structural requirements for cannabinoid activity we have been involved in the design and synthesis of stereochemically defined high affinity probes for the cannabinoid receptor. This effort has involved the development of irreversible ligands which will allow us to obtain detailed information on the cannabinoid receptor active site(s). The irreversible ligands, which incorporate highly reactive functional groups in a strategic position of the ligand, may form covalent bonds with amino acid residues at the receptor active site or in the neighborhood of this site. We shall discuss the biochemical properties of one of these probes, which incorporates the electrophilic isothiocyanate group into the structure of the highly potent cannabinoid agonist (-)-1',1'-dimethylheptyl-delta 8-THC. This ligand, (-)-7'-isothiocyanato-1',1'-dimethylheptyl-delta 8-THC (7'-NCS-DMH-delta 8-THC), was evaluated for its affinity for cannabinoid binding sites using rat forebrain membrane preparations and found to have an apparent IC50 value of 660 pM. Incubation of the membrane preparation with a ligand concentration of five times the apparent IC50 resulted in the irreversible occupation of nearly all of the receptor specific binding sites.

The synthesis of (2'RS)-2'-methyl-, (3'RS)-, (3'S)-3'-methyl-, and 4'-methyl-delta 8-THC has been carried out, and the pharmacology of all four compounds has been investigated. All four compounds showed typical cannabinoid activity both in vitro and in vivo. The 2'-methyl compound is somewhat more active than delta 8-THC, while the 4'-methyl isomer is less active. The 3'-methyl-delta 8-THC has approximately the same activity as the parent cannabinoid.

11-Hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol (1) was synthesized from the known cannabimimetic analog (+/-)-nabilone. Racemic 1 was resolved by HPLC on a semipreparative CHIRALCEL OD column (Daicel, Inc.), and pharmacological activities of the individual enantiomers were evaluated in the mouse model. The (-)-enantiomer was found to be much more potent than the (+)-enantiomer in all the four measures with the potency ratios in the production of catalepsy (RI), hypoactivity (SA), hypothermia (RT), and antinociception (TF) being 93, 143, 186, and 322, respectively. The racemic 11 alpha-OH diastereomer (2), a reaction side product, was also evaluated in the mouse model. Only small differences in the pharmacological activity of racemic 1 and 2 were found in the above four measures.

Delta-9-tetrahydrocannabinol ((-)delta 9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [3H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [3H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [3H]CP-55,940 to sperm, defined as total binding displaced by (-)delta 9THC, was saturable: KD 5.16 +/- 1.02 nM; Hill coefficient 0.98 +/- 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 +/- 122 cannabinoid receptors per cell. Binding of [3H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (-)delta 9THC, and (+)delta 9THC. The rank order of potency to inhibit binding of [3H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (-)delta 9THC > (+)delta 9THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [3H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation.

Small-angle neutron scattering was used to determine the intralamellar location of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) in hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers. Nuclear scattering density profiles were calculated from measurements on deuterium and non-deuterium-labelled inclusions (8.3% (w/w)) of delta 8-THC in DPPC multilayer samples. By comparing pairs of such nuclear density profiles, the locations of the deuterium labels were determined. Present results on the topography of delta 8-THC in membranes are compared with earlier X-ray measurements using iodine labelling. Whereas the position of the phenolic hydroxy group is similar in both types of measurement, a difference is found in the conformation of the terminal methyl groups of the cannabinoid side-chains. The X-ray measurements on dimyristoylphosphatidylcholine (DMPC) indicated that the iodine-labelled cannabinoid side-chains assume an all-trans orientation with the terminal iodine atom pointing inward into the membrane away from the tricyclic region while the neutron measurements indicate that the terminal CH3 group of delta 8-THC aligns itself at the level of the tricyclic ring system implying that the side chain exists in a more compact conformation perpendicular to the DPPC hydrocarbons. A Gaussian function analysis of the data indicates that the delta 8-THC molecule is significantly delocalized in the DPPC membrane in the liquid crystal phase. The mean location of delta 8-THC suggests that the active site on a membrane-embedded receptor protein will lie near the polar interface at the base of the phospholipid headgroups.

Small angle X-ray diffraction was used to study the topography of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) and its pharmacologically inactive methoxy analog (-)-O-methyl-delta 8-tetrahydrocannabinol (Me-delta 8-THC) in a membrane. The membrane preparations were partially hydrated dimyristoylphosphatidylcholine (DMPC) bilayers. Comparisons between electron density profiles from the drug-containing and drug-free membranes showed that the amphipathic delta 8-THC residues near the interface of the bilayer where the polar phenolic OH of the drug molecule is oriented towards the corresponding polar side of the phospholipid bilayer. Conversely, the highly hydrophobic Me-delta 8-THC distributes deeper in the bilayer away from the interface. Our results point out these two structurally-related, but pharmacologically very different, cannabinoids interact with membranes in strikingly different manners. This observation may, in part, explain the different pharmacological properties of the two cannabinoids.

Gas chromatographic-mass spectrometric methods of analysis for the detection of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid, a major metabolite of delta 9-tetrahydrocannabinol, are reviewed. Emphasis is on analytical methodology including numerous derivatization techniques developed specifically for this analyte. The majority of procedures cited in the literature were developed to detect this metabolite in the blood and urine of man.

The 1,1-dimethylheptyl (DMH) homologue of 7-hydroxy-delta 6-tetrahydrocannabinol (3) is the most potent cannabimimetic substance reported so far. Hydrogenation of 3 leads to a mixture of the epimers of 5'-(1,1-dimethylheptyl)-7-hydroxyhexahydrocannabinol or to either the equatorial (7) or to the axial epimer (8), depending on the catalysts and conditions used. Compound 7 discriminates for delta 1-THC (2) in pigeons (ED50 = 0.002 mg/kg, after 4.5 h), at the potency level of 3, and binds to the cannabinoid receptor with a KD of 45 pM, considerably lower than the Ki of 180 pM measured for compound 3 and the Ki of 2.0 nM measured for CP-55940 (1), a widely employed ligand. Tritiated 7 was used as a novel probe for the cannabinoid receptor.

delta 8-Tetrahydrocannabinol (delta 8-THC), a known antiglaucoma lipophilic drug, was incorporated in a submicron emulsion for ocular administration. The mean droplet size of the emulsion was 130 +/- 41 nm, and no droplet was larger than 400 nm. No change in pH, particle size distribution or zeta potential was noted after sterilization by steam autoclaving or long-term storage over 9 months. An intense and long-lasting intraocular pressure (IOP)-depressant effect was observed after ocular application (50 microliters) of the THC emulsion, 0.4% (w/w), to rabbits with ocular hypertension (chymotrypsin model). Lesser effects were observed in normotensive rabbits. No irritation effect of either the emulsion vehicle or THC emulsion on the rabbit eyes was detected. These results underline the promising properties of submicron emulsions as vehicles for lipophilic ophthalmic drugs. The mechanism by which the emulsion induced the marked delta 8-THC antiglaucoma effect remains unclear. However, the possible involvement of delta 8-THC systemic absorption in the hypotensive effect induced by the emulsion cannot be excluded and will be the subject of further investigation.

A brief history of the therapeutic uses and legal problems of cannabis as well as the component cannabinoids is given. This is followed by a discussion of drug development from delta 1-tetrahydrocannabinol and its synthetic analogues. The controversy of whether the pharmacological effects are of central or peripheral origin is included. Then, the potentials for the development of new drugs based on the cannabinoid structure for the treatment of pain, inflammation, and related conditions are outlined. It is concluded that the central activity of cannabinoids is confirmed and that the presence of a C-5 hydroxy group confers potent peripheral activity.

A novel [125I]-labelled photoaffinity ligand designed to detect cannabinoid binding sites has been used in mouse brain preparations and in cultured S49 mouse lymphoma cells. The ligand, 2-iodo-5'-azido-delta 8-THC, shows a high affinity for sites in both brain (Kd = 5.60 pM) and whole cell (Kd = 9.38 pM) systems. Photolabelling studies with brain samples revealed the existence of four ligand-protein adducts, of estimated molecular weights 85.5, 62.1, 30.0 and 25.5 kDa, that were diminished by prior exposure to 8 microM THC. A similar study with S49 cells gave adducts with apparent molecular weights of 62.1, 34.4, 16.9 and 13.5 kDa. The ligand produces a typical cannabinoid cataleptic response in mice suggesting that possibly one or more of the binding sites may be involved in some of the receptor mediated actions of THC.

The oxygenation of an aldehyde, 11-oxo-delta 8-tetrahydrocannabinol to a carboxylic acid, delta 8-tetrahydrocannabinol-11-oic acid was catalyzed by cytochrome P-450 MUT-2 purified from hepatic microsomes of male ddN mice. The oxygenation mechanism was confirmed by the incorporation of oxygen-18 from molecular oxygen into the carboxylic acid formed. An aldehyde form but not a hydrated form of 11-oxo-delta 8-tetrahydrocannabinol may be a substrate for the cytochrome P-450. The oxygenation of aldehyde catalyzed by cytochrome P-450 might be a common metabolic reaction in biological systems, and should be considered as an additional role of cytochrome P-450 in biotransformation of endogenous compounds and xenobiotics.

A simple liquid-liquid extraction and GC/MS-method for detection and quantification of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from merely 1 ml urine is described. The derivatisation to the methyl ester was carried out using methyl iodide and mefenamic acid was used as internal standard. Experiments with urine spiked with 15 ng THC-COOH/ml resulted in a recovery of 91%. Excellent linearity was obtained over the range 5-100 ng/ml.

delta 9-Tetrahydrocannabinol, the primary psychoactive constituent in marihuana, has been studied extensively for the last 20 years; however, the mechanisms responsible for cannabinoid activity in the central nervous system are not well understood. Although it is thought that lipophilicity plays an important role in the actions of cannabinoids, studies have not been conducted to determine whether a relationship exists between the lipophilicity and behavioral potency of cannabinoid analogs. Two procedures were used to obtain n-octanol/water partition coefficients (Po/w) of naturally occurring and synthetic cannabinoids: reverse-phase high-pressure liquid chromatographic estimation of Po/w and computer calculation of Po/w based on molecular structure. The Po/w value for delta 9-tetrahydrocannabinol obtained in this study, 9.44 x 10(6), is much greater than previously reported values obtained using shake-flask methodology, yet it is in agreement with the computer calculation based on molecular structure or molecular volume. The lipophilicity of the analogs determined in this study ranged from Po/w values of 3.92 x 10(2) to 1.93 x 10(11). All pharmacologically active cannabinoid compounds were extremely lipophilic (log Po/w values greater than 4.5). Several structural alterations were found to exert considerable influence on the lipophilicity of cannabinoid analogs. Increasing the length of the side chain in a homologous series of analogs results in an increase in lipophilicity of approximately 3-fold for each CH2 group added. Introduction of a single hydroxyl group decreased lipophilicity 3- to 40-fold, depending on the site of attachment. The behavioral potency of active analogs was not found to be correlated to lipophilicity. Therefore, data obtained in this study suggest that lipophilicity is a component, but not a primary determinant of pharmacological activity in the cannabinoids.

NMR and HPLC have been used to investigate the rearrangements of 1-O-acylglucuronides in vitro and the occurrence of rearranged isomers in urine. Glucuronides of flufenamic acid, (S)- and (R)-benoxaprofen, salicylic acid, and delta 9-11-carboxytetrahydrocannabinol were synthesized, by use of immobilized enzymes, or purified from urine. Ester-linked isomers of these gluruconides were characterized, and isomers derived from flufenamic acid, (S)-benoxaprofen, and salicylic acid were purified for further study by NMR and HPLC. The positions of the new ester linkages could be identified by two-dimensional NMR. Shifts not only in the resonance of the proton adjacent to the esterified hydroxyl group but also in the resonance of the anomeric proton on carbon 1 of the glucuronic acid moiety could be correlated with the position of each isomeric ester bond. HPLC elution times also correlated with ester position in this small set of samples. The sequences of isomer formation were studied in situ by NMR and also at pH 8 by HPLC. These studies indicate that, for the three cases examined, the C-2 ester is formed first, followed by formation of C-3 and C-4 esters. The purified isomeric esters were found not to re-form the high-energy 1-O-acyl bond. All other rearrangement steps are reversible. In contrast to other glycosides and glycerol esters, no evidence could be found for rearrangements beyond nearest-neighbor hydroxyl groups in glucuronic acid. The sequence of formation and reversibility is consistent with an ortho ester intermediate, as has been proposed for rearrangements of other glycosides.(ABSTRACT TRUNCATED AT 250 WORDS)