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We investigated the pharmacokinetic properties of D 9 -tetrahydrocannabinol (D 9 -THC), the main psychoactive constituent of cannabis, in adolescent and adult male mice. The drug was administered at logarithmically ascending doses (0.5, 1.6, and 5 mg/kg, i.p.) to pubertal adolescent (37-day-old) and adult (70day-old) mice. D 9 -THC and its first-pass metabolites-11hydroxy-D 9 -THC and 11-nor-9-carboxy-D 9 -THC (11-COOH-THC)-were quantified in plasma, brain, and white adipose tissue (WAT) using a validated isotope-dilution liquid chromatography/tandem mass spectrometry assay. D 9 -THC (5 mg/kg) reached 50% higher circulating concentration in adolescent mice than in adult mice. A similar age-dependent difference was observed in WAT. Conversely, 40%-60% lower brain concentrations and brain-to-plasma ratios for D 9 -THC and 50%-70% higher brain concentrations for D 9 -THC metabolites were measured in adolescent animals relative to adult animals. Liver microsomes from adolescent mice converted D 9 -THC into 11-COOH-THC twice as fast as adult microsomes. Moreover, the brains of adolescent mice contained higher mRNA levels of the multidrug transporter breast cancer resistance protein, which may extrude D 9 -THC from the brain, and higher mRNA levels of claudin-5, a protein that contributes to blood-brain barrier integrity. Finally, administration of D 9 -THC (5 mg/kg) reduced spontaneous locomotor activity in adult, but not adolescent, animals. The results reveal the existence of multiple differences in the distribution and metabolism of D 9 -THC between adolescent and adult male mice, which might influence the pharmacological response to the drug. SIGNIFICANCE STATEMENTAnimal studies suggest that adolescent exposure to D 9 -tetrahydrocannabinol (D 9 -THC), the intoxicating constituent of cannabis, causes persistent changes in brain function. These studies generally overlook the impact that age-dependent changes in the distribution and metabolism of the drug might exert on its pharmacological effects. This report provides a comparative analysis of the pharmacokinetic properties of D 9 -THC in adolescent and adult male mice and outlines multiple functionally significant dissimilarities in the distribution and metabolism of D 9 -THC between these two age groups.

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Background and Purpose: Fatty acid amide hydrolase (FAAH) is an intracellular serine amidase that terminates the signalling of various lipid messengers involved in pain regulation, including anandamide and palmitoylethanolamide. Here, we investigated the effects of pharmacological or genetic FAAH removal on tolerance to the antinociceptive effects of morphine. Experimental Approach: We induced tolerance in male and female mice by administering twice-daily morphine for 7 days while monitoring nociceptive thresholds by the tail immersion test. The globally active FAAH inhibitor URB597 (1 and 3 mgÁkg −1 , i.p.) or the peripherally restricted FAAH inhibitor URB937 (3 mgÁkg −1 , i.p.) were administered daily 30 min prior to morphine, alone or in combination with the cannabinoid CB 1 receptor antagonist AM251 (3 mgÁkg −1 , i.p.), the CB 2 receptor antagonist AM630 (3 mgÁkg −1 , i.p.), or the PPAR-α antagonist GW6471 (4 mgÁkg −1 , i.p.). Spinal levels of FAAH-regulated lipids were quantified by LC/MS-MS. Gene transcription was assessed by RT-qPCR. Key Results: URB597 prevented and reversed morphine tolerance in both male and female mice. This effect was mimicked by genetic FAAH deletion, but not by URB937. Treatment with AM630 suppressed, whereas treatment with AM251 or GW6471, attenuated the effects of URB597. Anandamide mobilization was enhanced in the spinal cord of morphine-tolerant mice. mRNA levels of the anandamide-producing enzyme N-acyl-phosphatidylethanolamine PLD (NAPE-PLD) and the palmitoylethanolamide receptor PPAR-α, but not those for CB 2 , CB 1 receptors or FAAH, were elevated in spinal cord Conclusion and Implications: FAAH-regulated lipid signalling in the CNS modulated opiate tolerance, suggesting FAAH as a potential target for opiate-sparing medications.

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)

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Effects of low estrogen combination type oral contraceptives on some of the biochemical parameters used for assessing vitamin nutritional status were investigated in a group of women who had used the pill for 6 to 12 months. Another group of women was examined initially and then at one or more points of time within the first 6 months of treatment. Following changes were observed in women treated with oral contraceptives: 1) increased excretion of kynurenic acid and xanthurenic acid following tryptophan load; 2) increased EGOT activity and also an increase in vitro stimulation of EGOT with added PALP; 3) increased plasma vitamin A levels; 4) fall in erythrocyte folate levels; 5) fall in erythrocyte transketolase activity with no change in vitro stimulation with TPP; and 6) fall in erythrocyte riboflavin concentration associated with a decrease in erythrocyte glutathione reductase activity and increase in vitro stimulation with FAD. Most of these changes were observed during the first few cycles of oral contraceptive treatment.