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MUNSON ET AL.
<br />fuged (1,000 rpm for 10 min) and washed twice in Dul-
<br />becco's medium containing 207. heat -inactivated fetal calf
<br />serum. They were then reconstituted to l01 cells/nd in
<br />Dulbecco's medium containing, for every 500 nil, 5 nal of
<br />200 mm glutamine, 5,000 U penicillin, and 5,000 pg strep.
<br />tomycin. Tumor cells (3–G ml) were dispensed into 25an1
<br />Erlenmeyer flasks and preincubated with either the drug
<br />or the drug vehicle for 15 minutes in a Dubnofl metabolic
<br />shaker at 370 C in an atmosphere of 5 Co. -95% O..
<br />After preincubation, 10 pl tritiated thymidine (1H -TDR)
<br />(10 pCi, 57 Ci/mmole; New England Nuclear Corp., Bos.
<br />ton, Mass.) was added to each flask and incubated for smri-
<br />ous times, after which Ia111 aliquots were removed -incl
<br />placed in 10x75.inm test tubes containing 1 1111 10 0
<br />trichloroacetic acid (TCA) at 4e C. The TCA-precipi-
<br />fated samples were then filtered on 0.•15-p Millipore fil.
<br />ters and washed twice with 5 ml of 10'. TCA at 4° C.
<br />True filters were transferred to liquid scintillation vials
<br />and counted in a toluene cocktail containing Liquifluor
<br />(New England Nuclear Corp.) (4 liters toluene to 160 nil
<br />Liquifluor). Samples were then counted in a liquid
<br />scintillator.
<br />Bone marrow: Bone marrow cells were derived from
<br />the tibias and fibulas of BDF, mice. One nil Dulbecco's
<br />medium containing 1 U heparin/ml was forced through
<br />each boric by n I -nil syringe with a 26.gauge needle. 7'he
<br />cells were washed three times, nucleated cells stere enu-
<br />merated on a henlocytonleter, and cell viability was ascer-
<br />tained by trypan blue exclusion. Cell number was ad-
<br />justed to 101 cells/ml with heparin -flee Dulbecco's
<br />medium mol incubated at 4e C for 15 minutes. ]tone
<br />marrow cells were then dispensed (3-5 ml) Into 25-m1
<br />Erlenmeyer flasks containing the lost drug or the drug
<br />vehicle. This preincubation period was followed by the
<br />addition of 10 pl 'H -TDR and the procedures done as
<br />outlined for the isolated Lewis lung cells.
<br />L1210: L1210 cells were derived from DBA/2 mice as
<br />described above. They were obtained from DBA/2 mice
<br />and inoculated 7 days before the experiment by the
<br />peritoneal cavity being flushed with 10 nil Dulbecco's
<br />medium containing heparin (5 p/ml). 7 -he cells were
<br />washed three times in medium, and the final medium
<br />wash dill not contain heparin. The cells were resus-
<br />pended at 101 cells/ml and treated as described above.
<br />Cells were routinely counted with a hemocytonleter for
<br />tire determination of cell viability with trypan blue; for
<br />Lewis lung tumor and L1210 cells, a Coulter apparatus
<br />(Mode ZB,) was also used.
<br />All other reagents were of the highest quality grade
<br />available. Actinomycin I), 5.Ouorouracii (5 -FU), and
<br />cytosine arabinoside (ara-C) were provided by the Drug
<br />Development Branch, National Cancer Institute (NCI).
<br />Cannabinoids.—The struc!ures of the four cmnpounds
<br />are shown in text -figure 1. All occur naturally in nrari.
<br />huana and were chemically synthesized. _Hicse drugs
<br />were provided by the National Institute on Drug Abuse
<br />or the Sheehan Institute for Research, Cambridge, Massa-
<br />chusetts. In clue preparation of the drugs, the cannabi.
<br />noids were complexed to albumin or solubilired in
<br />Enudphor-alcohol. Boih lrepnrations produced similar
<br />antitumor activity. With a�bunlin, the cannabinoids were
<br />prepared in the following planner: A stock solution of
<br />150 mg cannabinoid per ml absolute ethanol was made.
<br />Six mi of this solution was placed in a 200-m! flask. The
<br />ethanol was evaporated off under a stream of nitrogen
<br />and 2,100 mg lyophilized bovine serum albumin (LISA)
<br />added. After the addition of 20 ml distilled water, the
<br />CH3
<br />OH
<br />C5H I I (n )
<br />O9 -THC, Z -THC
<br />CH3
<br />� OH
<br />i
<br />CSHII(n)
<br />112 -THC, LI(6)-THC
<br />CH3 CH3
<br />\ OH \ OH
<br />� I i
<br />\ C5H11(n) \ \ I C5H11(n)
<br />OH
<br />Cannabinol (CBN) Cannabidiol (CBD)
<br />'rrxT-rrcear I.—SlraClllrer of lha four major canuabinoide.
<br />subswnces were stirred with a lass rod in a sonicator
<br />until a good suspension was achieved. Sufficient distilled
<br />water was then added to make the desired dilution. Con.
<br />centrations were routinely checked with a gas chromat.
<br />ograph. When Enndphor-alcohol was used as the velli.
<br />cle, the desired amount of cannabinoid was sonicated
<br />iu a solution of equal volumes by absolute ctluanol and
<br />Eniulphor (EI -620: GAF Corp., New York, N.Y.) and
<br />then diluted with 0.15 N NaCl for a final ratio of 1:1:4
<br />(ethanol: Ensulphor: NaCl).
<br />RESULTS
<br />Effects of Cannabinoids on Murine Tumors
<br />W- THC, As -THC, and cannabinol (CBN) all inhibited
<br />primary Lewis lung t unor growth, whereas cannabidiol
<br />(CBD) enhanced tumor growth. Oral administration of
<br />25, 50, or 100 mg Ar-THC/kg inhibited primary tumor
<br />growth by 48, 72, and 757t,, respectively, when measured
<br />12 days frost tumor inoculation (table I). On day 19,
<br />mice given Ar -THC had a 34% reduction in primary
<br />tumor size. On clay 30, primary tumor size was 76'/,that
<br />of controls and only those given 100 mgg Ar-THC/kg had
<br />a significant increase in survival time (36 ,).
<br />Mice treated with W -THC showed a slight weight loss
<br />over the 2 -week period (average loss. 0.3 g at 50 mg/kg
<br />and 0.1 g at 100 nig/kg). This can be compared to cyclo.
<br />phosphamide, which caused weight loss approaching 20'.
<br />(table 2).
<br />Al' -THC activity was similar 10 that of Ar -THC when
<br />administered orally daily until death (table 2). However,
<br />as with Ar -THC, primary tumor growth approached con-
<br />trol values after 3 weeks. When measured 12 days post
<br />tumor inoculation, all doses (50-400 mg/kg) of Aa-TIIG
<br />inhibited primary tumor growth between 40 and 60"6.
<br />Significant inhibition was also seen on day 21, which ara+
<br />comparable to cyclophosphamide -treated mice. Although
<br />this was not the optimum regimen for cyclophosphamide,
<br />it was the (>ositive control protocol provided by the NCI
<br />(1/). All mice given W -THC survived significantly longer
<br />than controls, except those treated with 100 mg/kg. Mice
<br />given 50, 200, and 400 mg/kg Ar -THC had an increased
<br />life -s an of 22.6, 24.6, and 27.2%, respectively, as corn*
<br />parol to 33;, for mice treated with 20 mg cyclophus-
<br />
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