A comparison of the disposition of 14C-Δ9-tetrahydrocannabinol and 3H-Δ9-tetrahydrocannabinol

Embed Size (px)

Text of A comparison of the disposition of 14C-Δ9-tetrahydrocannabinol and 3H-Δ9-tetrahydrocannabinol

  • Life Sciences, Vol . 21, pp . 441-450, 1977Printed In The U.S .A .


    Gordon T . Pryor*, Syed Husaint, and Albert J . Siemens**

    * Stanford Research Instltute, Menlo Park, California 94025, and** Research Institute on Alcoholism, Buffalo, New York 14203

    (Received in final form July 5, 1977)


    t University of North Dakota, Grand Forks, North Dakota 58202


    Pergamon Presa

    Preliminary experiments suggested that total levels ofradioactivity disappeared from the blood of male, Fischer ratsmuch morn rapidly following intragastric administration of14C-09 -tetrahydrocannabinoi ( 14C-THC) than 3H-THC . Collaborativeexperiments at the Stanford Research Instltute (SRI) and theResearch Institute on Alcoholism (RIA) verified and characterizedthe Initial observations . In rats that had food availablethroughout the experiments, the concentrations of total 3 H and14C in fresh plasma reached a maximum at 2 - 4 hours after treatmentwith 3H-THC plus 1`'C-THC . Thereafter, 14 C levels fell while 3Hlevels decreased very slowly or not at all . In fasted rats, peakplasma concentrations of both isotopes were not attained untilabout 8 hours following drug administration . The concentrationsof 14C then decreased more rapidly than 3F1 . The differencesbetween the plasma disappearance curves for 14C and 3H were notdependent upon the method of blood collection or the techniquesof isotope counting . However, when plasma or whole blood sampleswere dried before radioisotope analysis, the difference between1 `'C and ~I concentrations was virtually abolished to fed and fastedrats . Experiments suggest that tritiated water, produced duringthe metabolism of 3H-THC, may be responsible for the prolongedmaintenance of high 3H levels to the blood .

    Studies of the disposition of ~9 -tetrahydrocannabinoi (THC), the maJorpsychoactive component in cannabis, have been carried out to animals and manwith either 3H-THC or 14C-THC [see Bursteln (1) for review] . In many of theexperiments, body tissues and fluids were assayed for the unchanged drug aswell as for specific THC metabolites by various techniques of chromatography,mass spectrometry and liquid scintillation spectrometry . In addition, totallevels of 3F1 (2-8f or 1 `'C (9-15) were determined in numerous instances,providing Information on total drug equivalents . Evidently, these investiga-tors did not direct attention to the possibility that the two, differentlyradiolabelled THC molecules might not yield the same metabolic results .

    During studies of metabolic Interactions between THC and other psycho-active drugs at the Stanford Research Institute (SRI), tt was noted that thetotal concentration of Eli in the plasma of rats following 3H-THC administrationwas apparently greater than that of 14C following an equivalent dose of

  • 442

    Diaposition of 14C-A9-THC and ~-e9-THC

    vol . 21, No. 3, 1977

    14C-11C . A review of the results of experiments carried out with 3H-THC and1~C-THC at the Research Institute on Alcoholism (RIA) provided a similarobservation . It was considered important to investigate this observationmore fully because previously reported studies had apparently assumed that3F1- or 14C-THC would provide equivalent pharmacokinetic information . Further-more, other studies of THC/drug interactions at SRI and RIA required thisinformation to permit proper Interpretation of experimental results .

    THC Purity

    Unlabelled THC, 1`'C-THC* and 3H-THCt were provided by the NationalInstitute on Drug Abuse . Radiochemical purity of both labelled compounds wasgreater than 97$ as determined by thin-layer chromatography followed by radiochromatographlc scanning and liquid scintillation spectrometry (15) . Gaschromatography/mass spectrometry (Flnnigan, Model 1015D) using a 3$ OV17 Gas-Chrom Q 80/100 column demonstrated that the chemical purity of each compoundwas greater than 97$.

    Animals and Drug Administration


    Male Fischer rats (Simonsen Laboratories, Inc., G11roy, California),50 - 55 days of age at the time of the experiments and weighing 130-150 g,were used in all the experiments reported here. The animals were housedsingly in hanging stainless steal cages with food and tap water availablead libitum on a 12-12 hour light dark cycle for at least one week before eachexperiment . Food, but not water, was removed from half of the animals at1600 hours the night before the drug admlnistratlon and was withheld through-out the remainder of the experiment . The rest of the animals had free accessto food and water throughout the experimental period .

    The two radiolabelled compounds, 14C-THC and 3F1-THC, ware dissolved insesame oil together with unlabelled THC, resulting in a final total THCconcentration of 5 mg/ml with 20 uCi of 14C-THC/ml and 60 uCi of Eli-THC/ml .The drug solution was administered orally by gavage between 0800 and 0900hours to provide a total THC dose of 10 mg/kg . In some experiments, the ratswere irtmedlately placed into stainless steel metabolism cages to permitsequential urine collection .

    Assay of Radlolabelled TH Equivalents

    Blood samples (70 ul) were taken with heparlniznd micropipettes from theorbital sinus at both SRI and RIA at 1, 2, 4, 8, 12 and 24 hours after drugtreatment . In addition, blood was collected from the tall tip In another twogroups of fed and fasted rats at RIA at the same time points. Plasma (30 ul)from one sample was assayed directly in Oxifluor, New England Nuclear (SRI) orScintiverse, Fisher (RIA) by dual label counting (Beckman L5250 at SRI ;Packard 3385 at RIA) . Plasma from the other sample was combusted in aPackard 305 (SRI) or Packard 306 (RIA) Trlcarb Sample Oxidizer followed byliquid scintillation counting . Aliquots of urine samples ware also analyzedby combustion and liquid scintillation counting .

    * 14C was located at the 11-methyl position of the THC molecule .

    t 3H was located at the a and carbons of the pentyl side chain.

  • yol, 21, No . 3, 1977

    Diaposition of 14C-d9-THC and 3H-A9-THC


    All count data were corrected for quench and the concentrations of 3 F1-and 14C-THC equivalents were calculated based on the specific activities ofthe doses administered .

    Data obtained at RIA were computed and analyzed onthe PROPHET System, a unique computer resource of the National Institute ofHealth .


    In a series of preliminary experiments conducted at SRI, the oraladministration of either 1`'C-THC or 3H-THC resulted in maximum blood levelsof both isotopes at about 2 - 4 hours after treatment . Although 14 Cconcentrations declined thereafter, 3H levels continued to rise slightly upto 24 hours .

    Unlike the results obtained at SRI, two experiments at RIA felled to showany marked differences In the concentrations of 3H and 1 `'C in the blood ofrats over a 24-hr period following oral administration of 3H-THC or 1`'C-THC .Also, maximum levels of both Eli and 14C did not occur until about 8 to 12 hrafter drug treatment in the experiments at RIA, in contrast to the 2 - 4 hourpeak found at SRI .

    Discussion revealed that several differences existed In the protocolsused at SRI and RIA . At SRI the animals were allowed free access to foodthroughout the experiment and radioactivity was determined in whole blood orplasma samples suspended to liquid scintlllatton counting solution . At RIAthe animals were fasted overnight before the experiment and throughout thenext 24 hr and radioactivity was assayed after combustion of the bloodsamples (see "Methods" section) . In addition, blood had been collected fromthe orbital sinus at SRI and from the tall vein at RIA. In order to resolvethe discrepancies in the results and to sort out the relevant parameters,Independent experiments at SRI and RIA ware conducted using the same protocolsas described in the "Methods" section .

    Figure lA shows the results obtained at SRI by dual-label countingprocedures to nondeprived animals and demonstrates the basic differences inlaC and 3H concentrations over time that were observed initially in thepreliminary experiments . Concentrations of 14C rose rapidly to reach a clearmaximum after 2 - 4 hr and declined thereafter . Concentrations of 3H rose toa maximum after about the same time but fatted to decline significantlyup to 24 hr,

    Figure 18 shows the corresponding results in fasted animals and revealsone source of the difference between the results obtained at SRI and RIA notedabove. Instead of reaching maximum concentrations 2 - 4 hr after administration as in nondeprived animals, these maxima were not attained until about8 hr for both isotopes in the fasted animals . However, even though the maximawere delayed by 4 - 6 hr In the fasted animals the concentrations of 14Cdeclined thereafter, whereas the concentrations of 3H remained elevated overthe remainder of the 24-hr interval .

  • 444

    Diaposition of 14C-d9-THC and 3H-A9'-THC

    Vol. 21, No . 3, 1977








    0 .

    0 .4


    FIG . 1

    > I



    t12 4



    24 1 2 4




    Concentrations of 1 `'C ( " ) or 3 H (O) in the pertocular plasmaof fed (Panel A) or fasted (Panel B) rats determined by dual-label counting at SRI, Values are the means (t SE) from 15 ratsand are expressed as ug equivalents of D9-THC per milliliterof plasma.

    The SRI results for plasma shown to Figure 1 were reproduced in allessential aspects at RIA using the dual-label counting method of analysis,Figure 2A shows these results for fasted animals . The only slight differencesbetween the results in Figures 1B and 2A were in absol