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Munson 75.pdf


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fuged (1,000 r-pm for 10 min) and washed twice in Dulbecco’s medium containing 20% heat-inactivated fetal calf
serum. They were then reconstituted to LO7 cells/ml in
Dulbecco’s medium containing, for every 500 ml, 5 ml of
200 mM glutamine, 5,000 t_J penicillin, and 5,000 H streptomycin. Tumor cells (3-6 ml) were dispensed into 25-ml
Erlenmeyer flasks and preincubated with either the drug
or the drug vehicle for 15 minutes in a Dubnoff metabolic
shaker at 57” C in an atmosphere of 5% CO,-95% 08.
After preincubation, 10 ,,J tritiated thymidine (3H-TDR)
(10 &i, 57 CY/
I mmole; New England Nuclear Corp., Boston, Mass.) was added to each flask and incubated for various times, after which l-ml aliquots were removed and
placed in 10x75-mm test tubes containing 1 ml 10%
trichloroacetic acid (TCA) at 4O C. The TCA-precipitated samples were then filtered on 0.45-p Millipore filters and washed twice with 5 ml of 10% TCA at 4” C.
The filters were transferred to liquid scintillation vials
and counted in a toluene cocktail containing Liquifluor
(New England Nuclear Corp.) (4 liters toluene to 160 ml
Liquifluor). Samples were’ then counted in a liquid
scintillator.
Bone marrow: Bone marrow cells were derived from
the tibias and fibulas of BDF, mice. One ml Dulbecco’s
medium containing 1 U heparin/ml was forced through
each bone by a l-ml syringe with a 26-gauge needle. The
cells were washed three times, nucleated cells were enumerated on a hemocytometer, and cell viability was ascer_tained by trypan blue exclusion. Cell number was adjusted to 107 cells/ml with heparin-free Dulbecco’s
medium and incubated at 4’ C for 15 minutes. Bone
marrow cells were then dispensed (3-5 ml) into 25-ml
Erlenmeyer flasks containing the test drug or the drug
vehicle. This preincubation period was followed by the
addition of 10 ~1 3H-TDR and the procedures done as
outlined for the isolated Lewis lung cells.
L1210: L1210 cells were derived from DBA/2 mice as
described above. They were obtained from DBA/2 mice

and inoculated 7 days before the experiment by the
peritoneal cavity being flushed with 10 ml Dulbecco’s
medium containing heparin (5 p/ml). The cells were
washed three times in medium, and the final medium
wash did not contain heparin. The cells were resuspended at 10’; cells/ml and treated as described above.
Cells were routinely counted with a hemocytometer for
the determination of cell viability with trypan blue; for
Lewis lung tumor and L1210 cells, a Coulter apparatus
(Mode ZB,) was also used.
All other reagents were of the highest quality grade
available. Actinomycin D, 5-fluorouracil (5=FU), and
cytosine arabinoside (ara=C) were provided by the Drug
Development Branch, National Cancer Institute (NCI).
Cunnabinoids .-The structures of the four compounds
are shown in text-figure 1. All occur naturally in marihuana and were chemically synthesized. These drugs
were provided by the National Institute on Drug Abuse
or the Sheehan Institute for Research, Cambridge, Massachusetts. In the preparation of the drugs, the cannabinoids were complexed to albumin or solubilized in
Emulphor-alcohol. Both preparations produced similar
antitumor activity. With albumin, the cannabinoids were
prepared in the following manner: A stock solution of
150 mg cannabinoid per ml absolute ethanol was made.
Six ml of this solution was placed in a 200-ml flask. The
ethanol was evaporated off under a stream of nitrogen
and 2,100 mg lyophilized bovine serum albumin (BSA)
added. After the addition of 20 ml distilled water, the

A9 -THG,

A'

-THG

Gannabinol (GBN)

A*-THC, n”“-THC

Gannabidiol (GBD)

T EXT -FIGURE 1 .-Structures of the four major cannabinoids.

substances were stirred with a glass rod in a sonicator
until a good suspension was achieved. Sufficient distilled
water was then added to make the desired dilution. Concentrations were routinely checked with a gas chromatograph. When Emulphor-alcohol was used as the vehicle, the desired amount of cannabinoid was sonicated
in a solution of equal volumes by absolute ethanol and
Emulphor (El-620; GAF Corp., New York, N.Y.) and
then diluted with 0.15 N NaCl for a final ratio of I : 1:4
(ethanol : Emulphor : NaCl).
REWLTS
Effects of Cannabinoids on Murine Tumors

nS-THC, as=THC, and cannabinol (CBN) all inhibited
primary Lewis lung tumor growth, whereas cannabidiol
(CBD) enhanced tumor growth. Oral administration of
25, 50, or 100 mg a”-THC/kg inhibited primary tumor
growth by 48, 72, and 75.’%, respectively, when measured
12 days post tumor inoculation (table 1). On day 19,
mice given nS=THC had a 34% reduction in primary
tumor size. On day 30, primary tumor size was 76% that
of controls and only those given 100 mg A”-THC/kg had
a significant increase in survival time (36%).
Mice treated with A”-THC showed a slight weight loss
over the 2-week period (average loss, 0.3 g at 50 mg,/kg
and 0.1 g at 100 mg/kg). This can be compared to cyclophosphamide, which caused weight loss approaching 20%
(table 2).
A”-THC activity was similar to that of ag-THC when
administered orally daily until death (table 2). However,
as with A”-THC, primary tumor growth approached control values after 3 weeks. When measured 12 days post
tumor inoculation, all doses (SO-400 mg/kg) of A*-THC
inhibited primary tumor growth between 40 and 60%.
Significant inhibition was also seen on day 21, which was
comparable to cyclophosphamide-treated mice. Although
this was not the optimum regimen for cyclophosphamide,
it was the positive control protocol provided by the NC1
(II). All mice given as-THC survived significantly longer
than controls, except those treated with 100 mg/kg. Mice
given 50, 200, and 400 mg/kg a8-THC had an increased
life-span of 22.6, 24.6, and 27.2%, respectively, as compared to 33% for mice treated with 20 mg cyclophos-