|
s off • 7+tUNSON ET' A4
<br /> [aged (1,000 rpm for 10 min) and tva_~.ed twice in Dttl• CH3 CH3
<br /> Uecco's medium containing 20% heat inactivated fetal calf
<br /> scrum. They were then reconstituted to lOr cells/ntl in OH OH
<br /> DulUecco's medium containing, for every 500 mh 5 nil of
<br /> 200 mnt glutamine, 5,000 U penicillin, and 5.000 µg strep-
<br /> tomycin, Ttnnor cells (3-6 ml) wire dispensed into 25-nil CSHII(nl I C5H11(~)
<br /> Erlenmeyer Masks and preincubated with either the dntg
<br /> or the drug vehicle for 15 minutes in a Dubnoff metabolic g 1
<br /> shaker at 37° C in an atmosphere of 5 ~ COz 95% Os. ~ -THC, ~ THC Oe-THC, ~Itel-THC
<br /> Alter preincubmion, 10 µl triuated thymidine (rH•TDR)
<br /> (10 µCi, 57 Ci/mmole; New England Nuclear Corp., Bos-
<br /> ton, Afass.) x•as added to each Mask and incubated for vari•
<br /> ous times, after which 1•ml aliquots were removed and CH3 CH3
<br /> placed in IOX75•mm test tubes containing 1 ntl 10 ; ~ OH OH
<br /> trichloroacetic acid (TCA) at 4° C. The 1'CA•prccipi-
<br /> tated samples x•ere tjjten filtered on O,45•µ Alilliporc fil- ~
<br /> tars and x•ashed tx•ice with 5 ml of 10 ; TCA at 4° C.
<br /> The filters were transferred to liquid scintillation t•ials. I CgHtl(n) ~ C5H11(n)
<br /> and counted in a toluene cocktail containing Liquifluor OH
<br /> (New England Nuclear Corp.) (4 liters toluene to ICO ml
<br /> Liquilluor). Samples xere then counted in a liquid Connobinol (CBN) Cannabidiol (CBD)
<br /> scintillator.
<br /> 'irxr~ncvxr L-Strucwres of the tour major caunahinoids.
<br /> Bone marrox•: Bone marrow cells were derived from
<br /> the tiUias and fibulas of BDF, mice. One ml llulbecco's substances were stirred x•itlt a lass rod in a sonicator
<br /> medium containing 1 U heparin/ml teas forced through until a good suspension xas achtoved. Sufficient distilled
<br /> each bone by a 1•ml syrin with a 2f.gaugc needle. The
<br /> cells were washed three ut~»es, nucleatttl cells were enu• water was then added to make the desired dilution. Con•
<br /> meramd on a hemocytometer, and cell viability x•as aster- ~entrations were routinely checked with a gas chromat•
<br /> rained b}' tr}'pan blue exclusion. Cell number was ad- ograph. hen Emulphor-alcohol x•as used as the t•ehi•
<br /> jested to 10' cells/ml with he mrin•(ree DulUecco'a cle, the desired amount of cannaUinoid was sonicated
<br /> medium and incubated at 4° C ~or 15 minutes. (lone to a solution of equal volumes Uy abwlute ethanol and r
<br /> marrow cells were thth dispensed (3-5 ml) into 25•ml Emulphor (El•62U; GAF Cor New fork, 1~.Y.) and
<br /> Erlenmeyer flasks containing the teat drug or the drug then diluted x•ith 0.15 N NaC~ for a final ratio o[ 1: i :4
<br /> vehicle. This preincubation period ryas followed by tht (ethanal:Emulphor:laC1). i
<br /> addition of 10 µl aH•TDR and cite procedures done as RESULTS
<br /> outlined for the isolated Lewis lung cells.
<br /> L1210: L1210 cells were derived from DBA/2 mire as ERaets of CanmElnolds on Murln~ Tumor
<br /> described above. They were obtained from DBA/2 mice pr:
<br /> CHC, '•THC, and cannabinol (CBN) all inhibited
<br /> and inoculated 7 days before the experiment by the primar}• Lewis lung uunor grox•th, whereas cannabidiol
<br /> peritoneal cavity being Gushed with 10 ml DulUccco's (GBD) enhanced tumor growth. Oral administration of
<br /> medium containing heparin (5 µ/ml). The cells were 25, 511, or 100 mg Gr•THC(kg inhibited primary tumor
<br /> washed three times in medium, and the final medium growth by 48, 72. and 75 respectively-, when measured
<br />
<br /> ~ wash did not contain heparin. The cells were rases- 12 days jsost tumor inoculation (table 1). On day 19.
<br /> periled at IOr cells/ml and treated as described above. mice given A'•TfiC had a 34 ;redaction in primate
<br /> Cells were routinely counted with a hemocytometer for tumor sire. On clay 30, primary tumor sire x•as 7G a that
<br /> the determination of cell viability x•ith trypan blue; for of controls and only those gtven 100 mgg Gr-THC/kg had '
<br /> Lex•is lung tumor and L1210 «lls, a Coulter apparaws a significant increase in sun•ival time (36
<br /> (ttlode ZB,) was also used- Itlice treated with D°-THC showed a slight x•tight loss ;
<br /> All other reagents were of the highest qualityy grade over the 2•x•cck period (average loss, 0.3 g at 50 mg/kg
<br /> available. Actinomycin D, 5.Ouorouracil (5•FU), and and 0.1 g at 100 mg/kg). This can be compared to cyclo•
<br /> cytosine arabinoside (ara•C) were provided by the Drug phosphamide, which caused xeightloss approaching 20
<br /> Development Branch, National Cancer Institute (NCI). (table 2).
<br /> Canrmbinoids,-The structures-o[ the (our compounds A"-THC activity was similar [o that of ~r•THC when
<br /> are shoxas in text-figgure I. All occur naturally in mari• administered orally daily until Jeath (table 2). Hotvecer.
<br /> huana and were chemically synthesized. These drugs as with A'•THC. pnmary tumor growth approac)tecl con•
<br /> .were provided by dte National Institute on Drug Abuse trol values after 3 weeks. When measure) 12 days lzou
<br /> or the Sheehan Institute for Research, Cambridge, Itfassa• tumor inoculation. all doses (50-400 mg/kg) of A"•TI IC
<br /> chusetts. In the preparation of the drugs, the cannabi- inhibited primary tumor growth betx•cen 40 and GO'%~•
<br /> voids were complexed to albumin or solubilired in Significant mhibaron x•as also seen on day 21, which tra+
<br /> Enudphor-alcohol. Both (reparations produced similar comparable to cyclophosphamide-treated mice. Although I
<br /> antitumor activity. With albumin, the cannabinoids were this was not the optimum regimen for cyclophosphamidc•
<br /> prepared in the following manner: A stock solution o[ it was the (zositire control protocol provided Uy the NCI
<br /> 150 mg cannabinoid per ml absolute ethanol was mach (11). All mete given p"•TH survived significantly longer
<br /> Six ml of this solution x•as placed in a 200~m! Oask. The than controls, except those treater! with 100 mg/k8• Mire v
<br /> ethanol was evaporated o0 under a stream of nitrogen given 50, 200, and 400 mg/kg pr•THC had an mcreasecl
<br /> and 2.100 mg lyophilized Uovine scrum albumin ((SSA) life-span of 22.6. 24.11, and 27.21, respectivel}•, as com•
<br /> added. After the addition of 20 ml distilled water, the pared to 33 : for mice treated with 20 mg cyclophus•
<br />
<br />
|