Isotope effect studies on the dehydrogenation of Δ1-tetrahydrocannabinol in the rat

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<ul><li><p>1012 Specialia ]~XPERIENTIA 33/8 </p><p>The f inal concentrat ion of the non-radioact ive PgF2a was 0.1 ~zMoles NAD was appl ied as a co-factor in 4 mMoles f inal concentrat ion. The incubat ion period was 2.5 rain at 37 ~ under aerobic condit ions. The in tact PgF2a and the metabol i tes were dissociated by th in- layer chromato- graphy, and Packard Rad iochromatogram Scanner was appl ied to detect them (figure 1). The zones corresponding to PgF2~ and the metabol i tes were scarped and the radio- act iv i ty was measured w i th a Nuclear Chicaco L iquid Scint i l lat ion Spectrometer. The prote in content of the incubated substance was determined according to Lowry et al. ~3. Metabol ism of PgF2a is expressed according to Carminat i et al. ~ as pmoles of subst rate metabol ized h /mg protein. </p><p>400r </p><p>._ ~ 300 </p><p>200 </p><p>100 [ tMeao+-SO ~o, . . 0 ~ ' ' ~ ' ' ' ' r </p><p>5 6 1 8 9 10 11 12 15 14 Weeks of pregnancy </p><p>Fig. 2. Quantitative metabolism of PgF2a by human placenta during early pregnancy. </p><p>Results and discussion. The 15-OH-PGDH act iv i ty of the p lacenta shows a decl ining tendency from the 5 week of p regnancy ti l l the 9 week when a min imum can be observed, then they rise gradual ly as pregnancy advances (figure 2). P lacenta l prostag land in metabo l i sm per mg prote in was s igni f icant ly lower on week 9 of gestat ion (p &lt; 0.01 for week 9 then all other weeks). The exper imenta l results reported here demonst ra te that tkere is an impor tant re lat ionship between the stage of p regnancy and the metabo l i sm of PgFaa in early human placenta. That the p lacenta could be a r ich source of 15-OH-PGDH was demonst ra ted by Ja rabak 8, and par- t ial pur i f icat ions of th is enzyme from term placenta l t issue have been reported by Schlegel et al. ~4. I t can be suggested that the h igh enzyme act iv it ies in the p lacenta is par t of a mechan ism by which the fetus is protected against potent ia l ly harmfu l effects of h igh concentrat ions of prostaglandins. The results of our studies may indicate that decrease of 15-OH-PGDH act iv i ty can result in the rise in the endogenous prostag land in level, which makes a spontaneous contract ion of the uterus possible. The h ighest probab i l i ty of th is is in the f irst t r imester about the 9 week of the pregnancy. </p><p>13 O.H. Lowry, N. J. Rosenburgh, A. L. Farr and R. J. Randall, J. biol. Chem. 193, 265 (1951). </p><p>14 W. Sehlegel, M. Laurence, M. Demers, H. E. Hildebrandt-Stark, H. R. Behrman and R. O. Greep, Prostaglandins 5, 417 (1974), </p><p>Isotope effect studies on the dehydrogenation of A~'-tetrahydrocannabinol in the'rat </p><p>N. K. McCal lum, A. Guge lmann 1, C. A. M. Brenn inkmei jer 1 and R. Mechoulam 2, a </p><p>Chemistry Division, DSIR, Private Bag, Petone (New Zealand), 20 December 1976 </p><p>Summary. Isotope effect studies on the metabol ic dehydrogenat ion of A l - te t rahydrocannab ino l in rats are described and it is shown that this process is conf ined to a very short period fol lowing i.v. admin ist rat ion. The impl icat ions of this f inding are discussed. </p><p>A l though the pharmacologica l potency of Cannabis sa l iva can be a t t r ibuted main ly to its A l - tet rahydro - cannabino l (A~-THC) content ~, recent work does indicate that other cannabino ids such as cannabino l (CBN) can modify the effects of th is act ive const i tuent 5. Thus, observat ions that CBN accelerates clearance of A1-THC from the blood 6 and is also a rapid ly formed metabol i te of A1-THC 7, suggests that the metabol ic product ion of CBN may be involved w i th processes impor tant to cannab is intoxicat ion. </p><p>~2 OH O ~ </p><p>/I 1 -THC CBN </p><p>Since blood levels of metabol ica l ly produced CBN ~ are of l i tt le help in fol lowing i ts actual product ion it was decided to moni tor the metabo l i sm of A1-THC by uti l i - sat ion of the isotope effect. If t r i t ium is subst i tu ted for the C(3) proton (figure 1) and this p ro ton is involved in th is metabol ic process (as i t appears likely), the react ion rate will be slower s. Thus in a mixture of the subst i tu ted and unsubst i tu ted compound, a change in relat ive con- </p><p>centrat ion can provide evidence of react ion involv ing the hydrogen isotope; and the rate of change can provide a concent ra t ion - independent measure of the nature and d iscr iminat ion of th is part icu lar process. Unt r i t ia ted A1-THC was labelled wi th 14C in the aromat ic r ing so that relat ive concentrat ions of the t r i t ia ted and unt r i t ia ted species could be convenient ly moni tored by the rat io of the t r i t ium and 14C-activities. Materials and methods. A mixture of these label led com- pounds 9, unlabel led A1-THC and unlabel led CBN were puri f ied twice using preparat ive th in layer chromatog- raphy10,11. 3 studies were conducted. The f irst involved admin is t ra t ion of a mix ture of the 2 label led A1-THC species, tile second involved admin is t ra t ion of the 2 label led d l -THC species and unlabel led CBN, and tile th i rd involved admin is t ra t ion of the labelled A1-THC mixture to rats which had been pret reated twice dai ly for 5 admin is t rat ions of unlabel led zP-THC. The cannabino ids were admin is tered to rats 1~ in propylene glycol 13 as descr ibed previous lyL The A1-THC mixture was isolated f rom the blood 7 after fur ther unlabel led A 1-THC had been added, and puri f ied using th in layer chromatography 1~ Activies were determined by scint i l lat ion count ing and </p><p>3H cpm the rat ios were calculated 14 as I ' - - - and errors </p><p>14C cpm from count ing are quoted as lm Pure label led compounds were periodical ly subjected to the exper imenta l work-up dur ing the course of these exper iments, to conf i rm that </p></li><li><p>15. 8. 1977 Specialia 1013 </p><p>the ext rac t ion and separat ion had no in f luence on the f ina l rat io . Care was taken to ensure exper imenta l con- d i t ions were ident i ca l in al l deta i l s for compar i sons ~. Cont ro l s w i th the mix ture of the labe l led A~-THC spec ies be ing admin is te red , were run a t the same t ime as the exper iments out l ined in tab les 2 and 3 to check that compar i sons were val id . Results and discussion. I t has been demonst ra ted ~ that the product ion of CBN is not v ia non-metabo l i c dehydro - genat ion of A~-THC and we also conf i rmed that the method used in these exper iments was not respons ib le for any i so tope ra t io changes . I so tope ra t ios of the rad io - labe l led A1-THC iso la ted f rom the b lood a t d i f fer ing in terva ls a f te r in jec t ion of the labe l led A1-THC mixture are se t out ill tab le 1. I t wil l be noted that there is an apprec iab le change of ra t io less than 40 sec a f te r in jec t ion , but no s ign i f i cant t rend between the 40-see-and 28- ra in -sampl ings wh ich rep- resents 42 t imes the in i t ia l per iod ~5. Resu l t s f rom the hear t puncture sampl ing of a ra t a t 50 sec and fo l lowing decap i ta t ion at 3 rain were also the same wi th in count ing errors, and thus the t rans format ion of A1-THC wou ld appear to be tak ing p lace very rap id ly and p~ior to the 40-sec -sampl ing . Th is is in accordance w i th prev ious ana ly t i ca l data on metabo l i c CBN b lood levels in ra tsL The resu l t s c lear ly ind icate that we are observ ing an i so tope ef fect for the C(3) -H bond a l though it is not necessar i l y es tab l i shed that the a t tack is in i t ia l ly a t th i s pos i t ion . S ince it is known that cannab ino ids have an </p><p>3H cpm ) Table 1. Comparison of isotope ratios (I = 14C cpm of zJ1-THC </p><p>isolated from rat blood after varying intervals </p><p>Time elapsed I </p><p>0 sec 3.4 :~ 0.1 40 sec 4.7 0.2 50 sec* 4.2 0.4 3 min* 3.9 0.4 </p><p>10 min 4.9 ~ 0.2 28 min 4.8 4- 0.3 </p><p>* Sampled from the same rat. </p><p>Table 2. Isotope ratios after A1-THC administration with cold CBN </p><p>Time elapsed I </p><p>0 sec 3.4 =k 0.1 40 sec 3.82 i 0.03 3 min 4.9 i 0.15 7 min 4.2 ~ 0.15 </p><p>Table 3. Isotope ratios of A1-THC after administration to pretreated rats </p><p>Time elapsed I </p><p>0 sec 3.4 4- 0.1 40 sec (a) 5.4 0.15 40 sec (b) 5.0 4- 0.15 </p><p>ext remely h igh a f f in i tg for b lood pro te in 1~ the s imp les t exp lanat ion for the resu l t s in tab le 1 is that the advent of p ro te in b ind ing in the b lood is respons ib le for ha l t ing the progress of th i s react ion . Thus product ion of canna- b ino l imp l ies the presence of free A1-THC, and a com- par i son of ra tes of change of i so tope ra t ios a t d i f fe rent t imes enab les compar i son of the propor t ion of unbound or free A1-THC in the b lood at these t imes . I t has been suggested by O ldendor f that free d l -THC can be presumed to undergo complete c learance f rom the b lood by the bra in in a s ingle passage 1~, and it is known that w i th the more l ipophi l ic d rugs , rap id b ind ing to p lasma prote ins can reduce ava i lab i l i ty of the free drug for c ross ing the BBB and resu l t in an incomplete remova l of d rug to the bra in 1~, is. S ince pub l i shed repor ts , as wel l as th i s data for i so tope rat io changes , ind icate that the propor t ion of unbound A~-THC in the blood" a f te r 40 sec is ex t remely low 1~ and low levels of d rug (bound p lus unbound) also preva i l a f te r th i s t ime ~ the amount of free drug reach ing the bra in more than 40 sec a f te r the admin is t ra t ion wou ld appear to be </p><p>1 Institute of Nuclear Sciences, DSIR, Private Bag, Lower Hutt, New Zealand. </p><p>2 Department of Natural Products, School of Pharmacy, Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem, Israel. </p><p>3 The authors are grateful for generous gifts of 3H-A1-THC from Dr S. Burstein (Worcester Foundation for Experimental Biology, Massachusetts), and of 14C-A1-THC from the National Institute on Drug Abuse. The authors thank the staff of the Small Animal Unit, Wallaeeville Research Centre for their cooperation, and the U.S. National Institute on Drug Abuse for their support. </p><p>4 R. Meehoulam, A. Shani, H. Edery and Y. Grunfeld, Science 169, 611 (1970). </p><p>5 M. Fernandes, A. Schabarek, H. Coper and R. Hill, Psycho- pharmacologia 38, 329 (1974) ; R. N. Takahashi and I. G. Karniol, Psyehopharmacologia 47, 277 (1975); and cited references. </p><p>6 N .K . McCallum, Experientia 37, 957 (1975). 7 N .K . McCallum, B. Yagen, S. Levy and R. Mechoulam, Ex- </p><p>perientia 31, 520 (1975). 8 S. Aronoff, in: Techniques and Radiobiochemistry, p. 9. Iowa </p><p>State College Press, 1956. 9 8H-A1-THC labelled at C(3), was the kind gift of Dr S. Burstein </p><p>of the Worcester Foundation for Experimental Biology; x4C-A1- THC labelled at C(6') and C(4') in the aromatic ring was kindly supplied by Dr M. Braude of the National Institute on Drug Abuse, Maryland, USA. </p><p>10 J .A. Vinson and J. E. Hooyman, J. Chromatogr. 106, 196 (1975). 11 Purities of the canllabinoids were:zll-THC contained 0.4% CBN </p><p>by standard gle techniques and 0.1% CBN using varied ex- posures of a thin layer chromatogram to photographic plates; the CBN was 99.5% pure with ca. 0.1% s by glc. </p><p>12 Male Sprague-Dawley rats (130-150 g) were used with 1 mg CBN and/or 1 mg all, 14C-AI-THC (ca. 4 [xCi in total) per administration. </p><p>13 The propylene glycol showed a tendency to absorb moisture from the air causing the solution to form a cloudy, mierosus- pension of cannabinoid. Results using such a solution tended to be erratic (generally higher) unless the cannabinoid was solubilized by further warming or addition of traces of acetone and remixed. </p><p>14 Donald L. Horrocks, Applications of Liquid Scintillation Count- ing. Academic Press, New York and London 1974. </p><p>15 On the basis of blood level data 6 it can be argued that up to 35 sec after administration the injection plug is moving from the tail to the heart, and thus mixing (and therefore metabolism) can only take place after it reaches the heart, i.e. between 35 and 40 sec after administration. If this is the case the latter period represents not 42, but over 300 times the initial period during which metabolism is taking place. </p><p>16 E .R . Garrett and C. A. Hunt, J. pharm. Sci. 63, 1056 (1974). 17 W.H. Oldendorf, Proc. Soc. exp. Biol. Med. 147, 813 (1974). 18 A. Goldstein, L. Aronow and S. M. Calman, Principles of Drug </p><p>Action: The basis of Pharmacology, 2nd ed., p. 190. John Wiley &amp; Sons Inc., 1974. </p></li><li><p>1014 Specialia EXPERIENTIA 33/8 </p><p>minimal. The data also demonstrates -not unexpectedly - that relatively high proport ions of unbound molecules are available to the brain for an initial short period when high overall concentrat ions 7 of the drug prevail. This suggests that under these exper imental condit ions at least, the bulk of the A~-THC enters the brain within 40 sec of i.v. administrat ion. This shor t period encompasses the unstable s ituation prior to the equil ibration of free drug with blood proteins. Once inside the brain, avid binding of the Ai-THC to protein and membranes and possible slow metabol ism to 7-OH-Ai-THC ~9 would be expected to contr ibute to a slow release and prolonged duration of action~~ I t is possible that an enhancement of the t ransformat ion of A~-THC to CBN could be responsible for observed pharmacokinet ic interact ions between A~-THC and CBN% Table 2 i l lustrates the isotope ratios obtained from rats t reated with pure labelled A1-THC, and with labelled A~-THC and cold CBN under identical exper imental condit ions to those used above and ilo enhancement of the dehydrogenat ion of A1-THC is to be noted. Table 3 i l lustrates the results of another exper iment to simulate regular users; the rats were pretreated twice </p><p>daily for 5 administrat ions of unlabelled A~-THC before the pure labelled Ai-THC was finally administered. Again the difference does not appear to be sufficient to be the basis of differences in metabol ism rates ~t, but extended exper iments should be undertaken to confirm this. Thus the product ion of CBN from A~-THC in rats may be viewed as competit ion for available free Ai-THC, the effect of which on the overall intoxication, is yet to be earaluated. Enhancement of this t ransformat ion does not appear to be the basis of either the THC/CBN pharmaco- kinetic interact ion s or increased clearance rates of 'chronic users' 2i. </p><p>19 Pharmacology of Marihuana, p. 63. Ed. M. C. Braude and S. Szara. Raven Press, New York 1975. </p><p>20 Thiopental [A. Goldstein and L. Aronow, J. Pharmae. exp. Ther. 128, 1 (1960) provides an analogy with ZI1-THC. In this case lower lipophilicity results in less prote...</p></li></ul>