Metabolite of A9-THC

Like other xenobiotics, cannabinoids also undergo extensive metabolism in the human body to increase their hydrophihc properties for a facihtated ehmination. The metaboHsm of A9-THC has been very well investigated. More than 100 metabolites of A9-THC are known [99] and a good overview of the most important human metaboHtes is given in [100]. MetaboHsm takes place mainly in hepatic microsomes, but also in intestines, brain. 

The basic objective of this investigation was to establish sensitive methodology which would not depend on radio-labeling for the quantitative estimation of A9-THC, its primary metabolite 11-hydroxy-A9-THC (3), and cannabinol, which has been reported to be a metabolite of A9-THC in the rat (14). This objective has been realized, utilizing GLC-MS with a variety of techniques and instruments. In addition, the quantitative estimation of 11-nor-A9-THC-9-carboxylic acid has been accomplished. Several aspects of our results merit further discussion. 

The analysis for this "end" metabolite of A9 thc caused particular problems. Especially baffling for a considerable time was the analysis of 5a in plasma. 

This was interesting because there are several monohydroxylic metabolites of A9-THC. If we were monitoring, say, the molecular ion, we would detect all of these metabolites. Since 327 results from a loss of C-ll, this ion is specific for the derivative of ll-hydroxy-A9-THC. This is probably a minor point, because the various monohydroxylated metabolites have been shown to be separable by gas chromatography. Nevertheless, it does result in a highly specific determination for the most debated metabolite. 

Direct oxidation of A9-THC at position C-11 involves mainly an isomerization to A8-THC another opportimity in the synthesis of A9-THC-metabolites is the pretreatment of terpenoid synthons by introduction of protective groups, e.g., 1,3-dithiane (6.1 in Fig. 6) followed by the condensation with olivetol (6.2) [76]. The formed product is a protected derivate... 

A9-THC and its main metabolites are detected and quantified in forensic samples. Determination of these compounds in human beings is needed to make decision on abuse of A9-THC-containing drugs by individuals. A careful interpretation of the results is very important to avoid fallacies with regard to the behavior of individuals. The Cannabis influence factor (GIF), for example, is an useful tool for distinguishing between acute and chronic intake of A9-THC [98]. 

Pretreatment of hair samples also includes an extraction, usually with an alkaline sodium hydroxide solution, followed by cleaning up with LLE with n-hexane/ethyl acetate. Instead of LLE, the employment of SPE is also possible. Furthermore, the solid phase microextraction (SPME) in combination with head-space analysis is usable [104-106]. In the case of using hair samples, possible external contamination (e.g., by passive smoking of Cannabis) has to be considered as false positive result. False positive results can be avoided by washing of the hair samples previous to extraction [107]. Storage of collected samples is another important fact that can cause false results in their content of A9-THC and metabolites [108-110]. 

The application of GLC-MS techniques to the ill vitro and ill vivo metabolism of A9-THC (8) laid the groundwork for the quantitative analysis of other cannabinoid metabolites. The sites of metabolic hydroxylation for many cannabinoids are shown in Figure 1. 

A detailed discussion of many alternate methods for the quantitative determination of A9-THC and some of its metabolites has been presented previously (9). This paper will deal with determination of A9-THC, 11-hydroxy -A 9 -THC and cannabinol in blood with one eXtraC-... 

A plasma calibration curve for ll-nor-A9-THC-9-carboxylic acid, 5a, is shown in Figure 9. There was reasonable linearity from 1.0-50 ng/ml plasma with detection limits of 0.5 ng or less per ml. Figure 10 presents similar data for a urine calibration curve. The method showed reasonable linearity between 2.0-100 ng/ ml urine. Figure 11 presents pharmacokinetic data. for plasma levels of a human volunteer, BS, over a 0.5 hour to 48 hour period comparing A9-THC and 11-nor acid levels after a dose of 5.0 mg of A9-THC by the intravenous route. Both parent compound and acid metabolite exhibited a biphasic elimination pattern although the levels of the acid did not fall as rapidly as parent compound. Elimination of the acid metabolite 5a in urine is shown in Figure 12. It is evident that urinary elimination proceeded rapidly as 80% of the total 11-nor-acid excreted was eliminated in the urine during... 

The method would not permit separation of A9-THC from CBN, and in the case of 11-hydroxy- A9 thc, would not permit separation from other monohydroxy metabolites which might be present (8, 17). 

After 24 hours, 3-5 ng/ml of A9-THC were still found in the plasma. Our results for ll-hydroxy-A9-THC are probably the most accurate data yet reported in man. The concentration of this active metabolite (Figure 6) was only 2-3 ng/ml at peak levels declining at a slower rate than A9-THC to 0.5 ng/ml after 24 hours. Although A9-THC is readily converted to 11-hydroxy-A9-THC in the liver (3), only small quantities find their way into the blood. 

We present for the first time pharmacokinetic data in plasma and urine obtained by GLC-MS for the important acid metabolite 11-nor-A9-THC-9-carboxylic acid. This and related acids constitute the major means by which A9-THC is excreted in the urine. The data indicate rapid elimination of the acid in the urine during the first 3-6 hours after administration of A9-THC. 

Cannabinoid analytical work in our laboratory currently consists of two major areas of activity 1) quantitative analysis of A9-THC in samples of physiological fluids submitted by outside researchers under a NIDA program, and 2) development of an ultra-sensitive method for simultaneous quantitation of A9-THC and its major metabolites in body fluids. 

The cause for the differences has not been determined but these results prompted Mechoulam to warn that in the analysis of A9-THC and its metabolites it may not be possible to simply exchange plasma of different species but that each may, in fact, be different (3). 

The final analytical method for the simultaneous determination of A9-THC and its metabolites consists of the following sequence the cannabinoids are extracted from plasma with toluene they are then back extracted from toluene into Claisen s alkali the Claisen s alkali is diluted with water, tetrahexyl ammonium hydroxide is added and the alkaline solution is extracted with methylene chloride containing ethyl iodide. The overall recoveries were 45% for A9-THC and 83% for 11-hydroxy-A9-THC. External standards (l-0-ethyl-A9-THC and l-0-ethyl-ll-hydroxy-A9-THC) were added to the methylene chloride phase followed by a small amount of Florosil, which absorbed the tetra-hexylammonium hydroxide and tetrahexylammonium iodide. The methylene chloride was decanted and evaporated. 

The HPLC result reported here suggests a transfer of A9-THC into the breast milk of a chronic cannabis user. The calculated level of 0.26 yg/ml extrapolates to 26 yg per average 100 ml feeding which corresponds to 0.05-0.10% of the mother s estimated THC intake. It should be noted that this analysis was on a single sample and has not yet been confirmed by off-line spectral characterization. The identification of A9-THC in the milk must therefore be regarded as tentative. Future work requires multiple sample analyses from both average and chronic users. The milk should be analyzed for both A9-THC and metabolites. 

A9-THC is the major psychoactive constituent of Cannabis. Its detection and quantitation pose a difficult analytical problem because of its low concentration in biological fluids. Much work has been done on the identification and quantitation of A9-THC, its metabolites and cannabinoids by standard methods such as radio-immunoassay (1,2), gas chromatography, either alone (3-6) or coupled with mass spectrometry (7,8) and fluorometry (9-15). All these methods endeavor to satisfy two major criteria specificity and sensitivity. 

Our interest in CBN was aroused by reports from McCallum (14, 15) which indicate that CBN might be a transitory metabolite found at very early time periods after administration of A 9-THC. As shown in Figure 6, the level of CBN was below the reliability limits in the El mode. Other studies we have carried out by electron capture GLC or GLC-MS in the Cl mode indicate the virtual absence of this substance at all time periods. Since we have found that CBN has the same general pharmacokinetic pattern as A9-THC in man (4), we must conclude that CBN can be disregarded in terms of its importance as a metabolite in man. 

We attempted to approach the determination of ll-hydroxy-A9-THC in the same way. Preliminary experiments showed that ll-hydroxy-A9-THC was not very soluble in Brodie s solvent and the metabolite was unstable to methylation with trimethylanilinium hydroxide. 

We carried out a study in the dog to determine the formation of ll-hydroxy-A9-THC from A9-THC. In the first stage we injected 11-hydroxy-A9-THC in order to determine the beta phase half-life of the metabolite the half-life was approximately 1.5 hours. However, when A9-THC was administered to the same dog either orally or intravenously the metabolite was not detectable. 

Before entering into the study we felt that it would be useful to have a method that would determine both parent drug and metabolite simultaneously. There were two reasons for this first, the logistics of the work would be greatly improved since only one determination would have to be carried out per sample of plasma second, our original hypothesis was that, by exploitation of the chemistry of the phenol groups we could determine A9-THC and most of its metabolites. 

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