U.S. patent application number 09/851122 was filed with the patent office on 2002-02-21 for cannabinoid drugs.
Invention is credited to Burstein, Sumner H., Recht, Lawrence D., Zurier, Robert B..
Application Number | 20020022653 09/851122 |
Document ID | / |
Family ID | 22760084 |
Filed Date | 2002-02-21 |
United States Patent
Application |
20020022653 |
Kind Code |
A1 |
Burstein, Sumner H. ; et
al. |
February 21, 2002 |
Cannabinoid drugs
Abstract
The invention relates to the use of cannabinoid compounds
(derivatives of tetrahydrocannabinol) for decreasing cell
proliferation in a mammal.
Inventors: |
Burstein, Sumner H.;
(Framingham, MA) ; Recht, Lawrence D.; (Holden,
MA) ; Zurier, Robert B.; (Princeton, MA) |
Correspondence
Address: |
J. PETER FASSE
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Family ID: |
22760084 |
Appl. No.: |
09/851122 |
Filed: |
May 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60204935 |
May 17, 2000 |
|
|
|
Current U.S.
Class: |
514/454 |
Current CPC
Class: |
A61K 31/20 20130101;
A61K 31/35 20130101; A61P 43/00 20180101; A61K 31/19 20130101; A61P
35/00 20180101; A61K 31/353 20130101 |
Class at
Publication: |
514/454 |
International
Class: |
A61K 031/352 |
Claims
What is claimed is:
1. A method of decreasing cell proliferation in a mammal, the
method comprising identifying a mammal in which a decrease in cell
proliferation is desirable; and administering to the mammal a
compound having the formula 2wherein R.sup.1 is a hydrogen atom,
--COCH.sub.3, or --COCH.sub.2CH.sub.3; and R.sup.2 is a branched
C.sub.5--C.sub.12 alkyl, in an amount effective to decrease cell
proliferation in the mammal.
2. The method of claim 1, wherein R.sup.1 is hydrogen.
3. The method of claim 2, wherein R.sup.2 is a C.sub.9 alkyl.
4. The method of claim 3, wherein the C.sub.9 alkyl is a branched
alkyl.
5. The method of claim 4, wherein the branched alkyl is
1,1-dimethylheptyl.
6. The method of claim 1, wherein R.sup.2 is a C.sub.9 alkyl.
7. The method of claim 6, wherein the C.sub.9 alkyl is a branched
alkyl.
8. The method of claim 7, wherein the branched alkyl is
1,1-dimethylheptyl.
9. The method of claim 1, wherein the mammal is a human.
10. The method of claim 1, wherein the compound is administered
orally.
11. The method of claim 1, wherein the compound is administered
systemically.
12. The method of claim 1, wherein the compound is administered via
an implant.
13. The method of claim 12, wherein the implant provides slow
release of the compound.
14. The method of claim 1, wherein the compound is administered
intravenously.
15. The method of claim 1, wherein the cell proliferation is
associated with cancer.
16. The method of claim 1, wherein the amount of the compound
administered is about 0.1 to 20 mg/kg body weight of the
mammal.
17. The method of claim 16, wherein the amount of the compound
administered is about 0.2 to 2 mg/kg body weight of the mammal.
Description
[0001] CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority from U.S. Provisional
Application Ser. No. 60/204,935, filed May 17, 2000.
FIELD OF THE INVENTION
[0003] This invention relates to cancer therapy and organic
chemistry.
BACKGROUND OF THE INVENTION
[0004] The psychoactive agent in Cannabis plant material is
tetrahydrocannabinol (THC). Since THC is known to elicit various
physiological effects (e.g., as an anti-inflammatory agent or
analgesic) other than psychoactivity, various derivatives of THC
that retain a favorable biochemical or pharmacological activity of
THC without any potential for abuse or psychoactivity are
beneficial and have been synthesized as potential drugs.
[0005] One of the activities associated with THC and some of its
derivatives is inhibition of cell proliferation. However, this
activity, as with psychoactivity, is dependent on binding to the
cannabinoid receptor CB1 (Galve-Roperh et al., Nat. Med. 6:313-319,
2000; De Petrocellis et al., Proc. Natl. Acad. Sci. USA
95:8375-8380, 1998; and Bisogno et al., Eur. J. Biochem.
254:634-642, 1998). Thus, non-psychoactive derivatives of THC,
which do not bind to the CB1 receptor (Burstein, Pharmacol. Ther.
82:87-96, 1999), are not expected to inhibit cell
proliferation.
SUMMARY OF THE INVENTION
[0006] The invention is based on the discovery that
non-psychoactive THC derivatives, such as THC acids, can decrease
cell proliferation. Moreover, this effect is not dependent on an
increase in the rate of apoptosis, which has been identified as a
CB1 receptor-mediated activity of THC (Sanchez et al., FEBS Lett.
436:6-10, 1998).
[0007] Accordingly, the invention features a method of decreasing
cell proliferation in a mammal (e.g., a human) by identifying a
mammal in which a decrease in cell proliferation is desirable, and
administering to the mammal an amount of a compound of Formula I
effective to decrease cell proliferation in the mammal, 1
[0008] where R.sup.1 is a hydrogen atom, --COCH.sub.3, or
--COCH.sub.2CH.sub.3; and R.sup.2 is a branched C.sub.5--C.sub.12
alkyl. R.sup.1 can be hydrogen, and R.sup.2 can be a C.sub.9 alkyl,
which can be a branched alkyl such as 1,1-dimethylheptyl.
[0009] The compound can be administered orally (e.g., as a dietary
supplement), systemically, intravenously, or via an implant, which
can provide slow release of the compound. In addition, the compound
can be administered for treatment of a pre-existing disease or
condition that is characterized by cell proliferation, or for
prophylaxis against such a disease or condition. The compound can
be administered to the mammal at a dose of about 0.1 to 20 mg/kg
body weight (e.g., about 0.2 to 2 mg/kg body weight) to effectively
decrease cell proliferation. The method is particularly useful in
treating a mammal that is suffering from cancer.
[0010] In addition, a cell in vitro can be contacted with the
compound to decrease or abolish the cell's ability to
proliferate.
[0011] As used herein, "cell proliferation" means an increase in
cell number. By "decreasing cell proliferation" is meant a decrease
in cell proliferation that is not solely due to an increase in
apoptosis.
[0012] As used herein, "alkyl" means a straight or branched
hydrocarbon chain containing carbon atoms or cyclic hydrocarbon
moieties. These alkyl groups may also contain one or more double
bonds or triple bonds. By "substituted alkyl" is meant an alkyl in
which an atom of the alkyl is substituted with an atom, e.g., a
sulfur, oxygen, or halogen atom.
[0013] The methods of the invention provide a new use for
non-psychoactive cannabinoids as drugs for the treatment or
prophylaxis of a condition or disease characterized by cell
proliferations (e.g., cancer). Because of the low toxicity,
non-psychoactive nature, and low abuse potential of such
cannabinoids, the compounds can be used as a dietary supplement
(e.g., like a daily vitamin pill) to prevent cancer. In addition,
the compounds can be applied topically, e.g., to a skin lesion
characterized by undesirable cell proliferation, such as in
psoriasis.
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
suitable methods and materials for the practice or testing of the
present invention are described below, other methods and materials
similar or equivalent to those described herein, which are well
known in the art, can also be used. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0015] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a line graph of concentration of various compounds
versus cell number.
[0017] FIG. 2 is a bar graph of ajulemic acid concentration versus
cell number.
[0018] FIG. 3 is a line graph of concentration of various compounds
versus neutral lipids per million cells.
[0019] FIG. 4 is a line graph of days post-inoculation versus tumor
diameter.
DETAILED DESCRIPTION
[0020] The invention relates to methods of decreasing cell
proliferation (e.g., cancer treatment) in a mammal by administering
a THC derivative to the mammal. These THC derivatives (e.g., the
compounds defined by Formula I) have reduced or no psychoactivity
and do not bind to the CB1 receptor. Such THC derivatives are known
and can be synthesized (see, e.g., U.S. Pat. No. 5,338,753;
Burstein et al., J. Medicinal Chem. 35:3185-3141, 1992; and
Burstein, Pharnacol. Ther. 82:87-96, 1999).
[0021] The THC derivative can be administered via any appropriate
route, e.g. intravenously, intraarterially, topically, by
injection, intraperitoneally, intrapleurally, orally,
subcutaneously, intramuscularly, sublingually, intraepidermally, or
rectally. It can be formulated as a solution, suspension,
suppository, tablet, granules, powder, capsules, ointment, or
cream. In the preparation of these pharmaceuticals, a solvent
(e.g., water or physiological saline), solubilizing agent (e.g.,
ethanol, Polysorbates, or Cremophor EL7), agent for making
isotonicity, preservative, antioxidizing agent, excipient (e.g.,
lactose, starch, crystalline cellulose, mannitol, maltose, calcium
hydrogen phosphate, light silicic acid anhydride, or calcium
carbonate), binder (e.g., starch, polyvinylpyrrolidone,
hydroxypropyl cellulose, ethyl cellulose, carboxy methyl cellulose,
or gum arabic), lubricant (e.g., magnesium stearate, talc, or
hardened oils), or stabilizer (e.g., lactose, mannitol, maltose,
polysorbates, macrogols, or polyoxyethylene hardened castor oils)
can be added. If necessary, glycerin, dimethylacetamide, 70% sodium
lactate, a surfactant, or a basic substance such as sodium
hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate,
arginine, meglumine, or trisaminomethane is added. Pharmaceutical
preparations such as solutions, tablets, granules or capsules can
be formed with these components. Compositions for slow release of
the compound can be formed as described in U.S. Pat. No.
4,880,830.
[0022] The dose of the compounds of the present invention is
determined in consideration of the results of animal experiments
and various conditions. More specific doses obviously vary
depending on the administration method, the condition of the
subject such as age, body weight, sex, sensitivity, food eaten,
dosage intervals, medicines administered in combination, and the
source, seriousness, and degree of the affliction. The optimal dose
and the administration frequency under a given condition must be
determined by the appropriate dosage test of a medical specialist
based on the aforementioned guide.
[0023] Before administration into humans, THC derivatives can be
tested for biological activity (i.e., ability to decrease cell
proliferation) both in vitro or in vivo. In vitro testing can be
performed as described in the example below, as well as described
in Marshall et al., Growth Reg. 5:69-84, 1995. In vivo animal
models for tumor growth are well known, such as described in Nagane
et al., Cancer Res. 60:847-53, 2000; and Price et al., Clin. Cancer
Res. 5:845-54, 1999.
[0024] The invention will be further described in the following
example, which does not limit the scope of the invention defined by
the claims. The example below illustrates the use of
1',1'-dimethylheptyl-.DELTA..sup- .8-THC-11-oic acid (also known as
CT3 and ajulemic acid) in decreasing cell proliferation.
EXAMPLE
[0025] Materials and Methods
[0026] Cells and materials. C6 glioma and U87 glioma cells were
obtained from ATCC (Manassas, Va.). Other human lines were
harvested and maintained in the Cancer Center Tumor Bank. WI38
cells were obtained from the Tissue Culture Facility of the
University of Massachusetts Medical Center (Worcester, Mass.). All
chemicals and solvents were obtained from Sigma-Aldrich (St. Louis,
Mo.) unless indicated otherwise. THC and THC-11-oic acid were
supplied by NIDA. Ajulemic acid was synthesized as described in
Burstein et al., J. Medicinal Chem. 35:3185-3141, 1992.
[0027] Cell proliferation assays. In vitro changes in the number of
cells were measured using the MTT assay as described in Marshall et
al., Growth Reg. 5:69-84, 1995.
[0028] Flow cytometry/cell cycle analysis. For flow cytometry,
cells were grown in low serum (0.5%) for two days to synchronize
cells in G0/G1, after which they were placed in high serum (10%)
for two days. The cells were then labeled with propidium iodide and
fixed in 4% paraformaldehyde. Cells were examined using a MoFlo
Facs sorter for DNA content.
[0029] Lipid incorporation assay. Cells were treated as follows.
Monolayers of C-6 cells were prepared in 24 well culture dishes as
described above. Carboxy labelled .sup.14C-arachidonic acid
(150,000 dpm/well) obtained from ARC, St. Louis, Mo. (specific
activity of 55 mCi/mmol) was added to each monolayer and incubated
for 2 hours. Treatment with the indicated cannabinoid was initiated
by the addition of the drug in 10 .mu.l of DMSO to 1 ml of the
culture medium covering each monolayer. Treatment was continued for
48 hours, except as indicated otherwise. After this incubation, the
media were removed and discarded. After washing twice, each time
with 1 ml PBS, the cellular lipids were extracted for 1 hour with
0.5 ml of absolute ethanol at room temperature. All treatments were
performed in quadruplicate. Controls cells were treated
identically, except that no cannabinoid was present.
[0030] To perform group analysis of the extracted lipids, the lipid
samples were lyophilized. Prior to evaporation under vacuum,
.sup.14C-cholesterol (50,000 dpm) was added as a recovery marker
(ARC, St. Louis, Mo.; specific activity of 50 mCi/mmol). The sample
residues were then dissolved in 30 .mu.l of methanol containing 10
.mu.g each of steroyl-arachidonoyl diglyceride, triolein, and
lecithin and applied to 0.25 mm silica gel thin layer plates. A
first elution was performed using a 9:1 mixture of
dichloromethane:acetone for analysis of neutral lipids. The Rf
values of the standards were as follows: lecithin=0,
cholesterol=0.38, diglyceride=0.64, and triglyceride=0.81.
Following the quantitation of the neutral lipids, a second elution
was carried out using a 50:25:8:4 mixture of
chloroform:methanol:acetic acid:water as the eluent for the
analysis of phospholipids. The Rf value of lecithin was 0.33. DG
and TG moved to the solvent front. All standards were detected by
exposure to iodine vapor.
[0031] Labelled lipids were quantitated as follows. The zones of
radioactivity were detected by exposure of the plates to X-ray film
for 48 hours. A TIFF computer file of the film was generated using
the Fluor-S System (Biorad). The chromatograms were quantified
using NIH Image software. Peak height values of the display were
used, since all labelled components resided in narrow, sharp peaks
on the chromatograms. Each component value was adjusted for
recovery using the individual cholesterol standard values for each
zone. The values obtained were then divided by the numbers of cells
in each well and the results expressed as an index per million
cells.
[0032] Protein kinase C (PKC) assay. Cells were transferred into
MEM media (0.5% serum) and cultured overnight. The next day, cells
were treated with either DMSO or 25 .mu.M ajulemic acid for the
specified incubation time, after which the plates were trypsinized.
5.times.10.sup.6 trypsinized cells were assayed for PKC activity
using a kit (Calbiochem, cat. no. 53984).
[0033] In vivo subcutaneous model. 10.sup.6 U87 cells were injected
into the right flank muscle of male nu/nu BALBc mice (Charles
River). Two days after inoculation, the mice received either 0.1
mg/kg ajulemic acid in 0.05 ml of safflower oil, or 0.05 ml
safflower oil by oral administration. Thereafter, identical dosings
were performed on each Monday, Wednesday, and Friday. When visible
tumors appeared, the size of the tumors were estimated by averaging
the values of two roughly perpendicular diameters measurements.
Visible tumors were measured on the days of drug
administration.
[0034] Results
[0035] Using the MTT assay to assess growth potential, it was noted
that .DELTA..sup.9-THC, THC-11-oic acid and ajulemic acid all
inhibited C6 glioma cells in a dose dependent fashion with
IC.sub.50 values of 10, 20 and 55 .mu.M, respectively (FIG. 1). The
proliferation of a number of human cell lines derived from a
variety of cancer types (brain, breast, bladder, lung, and
prostate) was tested for sensitivity to 25 .mu.M of each agent for
48 hours. At this dosage, ajulemic acid inhibited cell
proliferation better than either THC or THC-11-oic acid. (As used
herein, "THC" refers to .DELTA..sup.8-or .DELTA..sup.9-THC.) The
inhibition levels were 61.3.+-.12.1, 39.4.+-.22.0, and
13.1.+-.18.5% (mean.+-.SD) for ajulemic acid, THC, and THC-11-oic
acid, respectively, with p<0.001 in a one-way ANOVA, pairwise
multiple comparison (Student-Newman-Keuls method), as shown in
Table 1.
1TABLE 1 Percent inhibition of human cancer cell line growth in 48
hour MTT assay Tissue of .DELTA..sup.9-THC Ajulemic Acid THC-11-oic
acid Line origin (25 .mu.M) (25 .mu.M) (25 .mu.M) U87 Brain 20 .+-.
5 54 .+-. 2 5 .+-. 1 U373 Brain 0 34 .+-. 1 0 U118 Brain 39 .+-. 5
60 .+-. 2 14 .+-. 3 A172 Brain 42 .+-. 5 76 .+-. 1 61 .+-. 2 HS578T
Breast 52 .+-. 5 60 .+-. 2 2 .+-. 1 HT1376 Bladder 23 .+-. 5 60
.+-. 2 21 .+-. 12 J82 Bladder 63 .+-. 6 57 .+-. 3 1 .+-. 1 Calu6
Lung 28 .+-. 5 69 .+-. 3 19 .+-. 5 Du145 Prostate 55 .+-. 7 74 .+-.
2 7 .+-. 3 PC3 Prostate 72 .+-. 2 69 .+-. 2 1 .+-. 1
[0036] Compared to THC, ajulemic acid was significantly more
effective (p<0.05) at this dosage in six of the ten lines
tested.
[0037] The three cannabinoids also inhibited the normal rat
fibroblast cell line WI38. However, it was notable that, unlike
THC, ajulemic acid inhibited the proliferation of WI38 cells less
than the proliferation of C6 glioma cells. For example, in three
different experiments at the 25 .mu.M dosage, .DELTA..sup.9-THC
inhibited W138 cell proliferation 66%, in comparison to 50% for the
C6 cells. By contrast, incubation with ajulemic acid for two days
resulted in an opposite effect. Thus, there was a 65% inhibition
for C6 compared to 44% for WI38 cells. The data suggests that
ajulemic acid was more toxic to tumor than to normal cells.
[0038] To assess whether ajulemic acid's effect was
receptor-mediated, a preparation 95% enriched in the D-isomer of
ajulemic acid was used to examine the stereospecificity of the
effect observed above. There was a marked decrease in the potency
of a 25 .mu.M dose of the D-isomer compared to the L-isomer of
ajulemic acid (p<0.001, t-test) (FIG. 2). This result supported
the contention that ajulemic acid's activity was stereospecific and
therefore most likely receptor-mediated.
[0039] Three features of ajulemic acid's effect on tumor cells
suggested that this effect was mediated by a mechanism distinct
from that of .DELTA..sup.9-THC. First, the compound's inhibition of
cell proliferation was not reversible with a CB1 antagonist.
Co-incubation of C6 glioma cells with 3.2 .mu.M of a specific
antagonist resulted in a 150% decrease in the potency of THC,
compared to a slight potentiation of ajulemic acid's effect (FIG.
3). This result indicates that a large component of THC's antitumor
effect is CB1 receptor-mediated, while ajulemic acid's were
not.
[0040] Second, ajulemic acid's antitumor activity was associated
with a marked increase in cell size after 48 hours of exposure to
the compound. In order to investigate this effect further, the
effect of ajulemic acid on cell cycle kinetics was assessed. C6
glioma cells were incubated for 24 hours in media containing 0.5%
serum to synchronize them in G1/G0. The cells were then exposed to
media containing 10% serum either with or without 25 .mu.M ajulemic
acid for two days. As shown in Table 2, an increased number of
treated cells was seen in both S (from 4.4% to 13.0%, p=0.003,
t-test) and G2/M (from 21.9% to 32.3%, p=0.005, t-test) phases of
the cell cycle, suggesting that treated cells could synthesize DNA
but were not able to complete mitosis. Importantly, the rate of
apoptosis (as assessed by the lack of sub-G0 cells in both control
and treated cells) was not increased.
2TABLE 2 Cell cycle analysis of Control and AJA-treated C6 glioma
cells G0/G1 S G2/M (mean % .+-. SD) (mean % .+-. SD) (mean % .+-.
SD) Control 48.3 .+-. 3.6 4.4 .+-. 0.3 21.9 .+-. 1.1 AJA-treated
39.1 .+-. 1.3* 13.0 .+-. 2.3** 32.3 .+-. 3.0*** *p < 0.02, **p =
0.003, and ***p = 0.005, all determined by t-test
[0041] Third, another distinctive feature of ajulemic acid's
antitumor effect was the appearance of refractile bodies that could
be easily visualized with phase microscopy. Both the number of
cells that had refractile bodies as well as the number of
refractile bodies/cell increased over time. After 48 hours, 83% of
ajulemic acid-treated C6 cells had one or more refractile bodies
compared to 27% for THC-treated cells (p<0.001, Chi-square
analysis). The mean number of refractile bodies per cell was also
much higher for ajulemic acid-treated cells compared to THC-treated
cells (1.78.+-.1.6 vs. 0.22.+-.0.5 per cell, p<0.001, t-test).
Interestingly, no refractile bodies were noted in either W138 cells
treated for this length of time at this dosage of ajulemic acid or
in C6 glioma cells treated with 25 .mu.M THC-11-oic acid.
[0042] To further explore the nature of the cell enlargement and
refractile bodies, cells were examined under electron microscopy.
Ajulemic acid-treated cells were neither apoptotic nor necrotic. In
fact, these cells appeared metabolically active. This is consistent
with additional observations that, although cell proliferation was
markedly inhibited by ajulemic acid, the treated cells have a high
(>95%) viability rate as assessed by trypan blue exclusion and
recover quickly upon drug removal. Moreover, ajulemic acid-treated
cells were enlarged and contained large lipid droplets, the
identity of which was confirmed by bleaching with osmium
tetrachloride.
[0043] To investigate the source of the increased lipid content, C6
cells were grown and treated with 25 .mu.M dose of cannabinoid in
the presence of .sup.14C-labeled arachidonic acid. Compared to
cells treated with vehicle (no cannabinoid), THC, or THC-11-oic
acid, a significant increase above basal levels was only noted for
ajulemic acid (p<0.05, Dunnett's test of multiple comparisons
versus control) after 48 hours of incubation (FIG. 3). At this dose
of ajulemic acid, the content of both tri-and di-glyceride was over
three-fold greater than in controls. By contrast, no significant
increase in the level of phosphatidylcholine for any of the
cannabinoid treatments was detected. A similar result was seen
after incubation with a .sup.14C-oleic acid label.
[0044] Because diacyl glycerol (DAG) is an important signal
transducer of protein kinase C, the effect of ajulemic acid
treatment on the PKC activity in C6 cells was examined. After
exposure to ajulemic acid, PKC activity increased within five
minutes and remained approximately two-fold above that of control
for one hour, suggesting that PKC plays a role in ajulemic
acid-mediated inhibition of cell proliferation. At least two
alternatives explanations for this observation exists: (1) the PKC
isozymes that were stimulated were those which slow growth, such as
PKC.delta.; and (2) the increased DAG was modulating some other
downstream mediator, such as .beta.-chimaerin, to a greater extent
such that the balance is towards anti-proliferation.
[0045] Whether ajulemic acid has in vivo antitumor activity was
determined using a nude mouse tumor model. The growth of the human
glioma cell line U87 injected subcutaneously in nude mice was
assessed with and without ajulemic acid administration. For these
experiments, ajulemic acid treatment was initiated two days after
inoculation of 10.sup.6 tumor cells in the mice. A delay in both
the appearance and size of tumors were seen for the group receiving
0.1 mg/kg ajulemic acid thrice weekly (FIG. 4). At day 25
post-inoculation, 5 of 5 control mice had tumors with a mean
diameter of 16.3.+-.3.2 mm, while the mean diameter in the treated
group was 3.8.+-.8.5 mm (n=5, p=0.015, t-test). These in vivo
results confirm the ability of ajulemic acid to inhibit cell
proliferation, in this case in a tumor.
* * * * *