Method for treating human tumor cell metastasis

A therapeutic method for reducing metastasis is disclosed. The method involves administering a therapeutically effective amount of 3-methyl-1-[2-(2-naphthyloxy)-ethyl]-2-pyrazolin-5-one or a pharmaceutically acceptable salt.

BACKGROUND AND PRIOR ART 
The primary goal of cancer treatment is treatment and eradication of the 
growth of the primary tumor. Concurrent with this treatment it is 
necessary to prevent metastasis, which can be defined as separation of 
primary tumor cells and their subsequent penetration into the lymphatic 
system or blood vessels for dissemination. Such dissemination may occur by 
adhesion to and subsequent penetration through the endothelial walls, 
establishment of secondary tumors in the perivascular tissues and eventual 
spread of the tumor cells to more distant sites. Although much is known 
about the clinical manifestations of the metastatic process, little is 
understood about the biochemical, immunologic, genetic, and hormonal 
mechanisms involved in metastasis. Thus metastasis can be considered as a 
single phenomenom represented by an intricate series of events. 
Because of the importance of both treatment of primary tumor growth and 
prevention of metastasis, cancer researchers have undertaken extensive 
research to define the interactions involved in tumor growth and 
metastasis. 
One of the biological properties which tumor cells appear to possess is the 
ability to interact with and to attach to host blood platelets, enhancing 
the potential of the tumor to lodge in the microvasculature and adhere to 
vascular endothelium. Alternatively, it has been suggested that following 
lodging of the tumor cells, the cells may initiate the formation of 
surrounding protective platelet thrombi. For successful metastasis to 
occur, the metastatic cells must first lodge and adhere to the vascular 
endothelium and remain intravascular until it infiltrates into the 
surrounding tissue. 
Because of the similarities of the process involved in the lodging and 
adherance of the tumor cells to the endothelium and the formation of 
non-tumor thrombi, many investigators have concluded that platelets are 
involved in some fashion. Because of this platelet involvement, numerous 
investigations have been undertaken to determine the effect of 
anticoagulant therapy on metastasis. The investigations referred to below 
involved the administration of anticoagulant compounds which are potent 
inhibitors of platelet aggregation. The results to date have been 
ambivalent. 
Heparin has been reported to both decrease and increase metastasis, 
especially pulmonary. [See Cell Biol. Intl. Rep. 2: 81-86 (1963) and Arch. 
Surg. 91: 625-629 (1965)]. Aspirin has produced mixed results [See Eur. J. 
Cancer 8: 347-352 (1972) and Intl. J. Cancer 11: 704-718 (1973)]. Warfarin 
has been demonstrated to produce significant antimetastatic effects after 
intervenous injection of tumor cells and in spontaneously metastasizing 
tumors [See Cancer 35: 5-14 (1975) and Cancer Res 37: 272-277 (1971)]. It 
has been shown that metastasis induced by intravenous administration of 
B-16.sub.a melanoma cells can be prevented by administration of the 
anticoagulant agent prostacyclin [See Cell Biol. 87: 649 (1980)]. 
For a review of the use of anticoagulants in tumor therapy, see M. B. 
Donati, et al., Malignancy and the Hemostatic System, pp. 103-120, Raven 
Press, 1981. 
It has been suggested that the use of anticoagulant therapy has been less 
than satisfactory in part because of the lack of specificity of the 
anticoagulant agents used and the fact that some of the agents produce 
effects on the tumor cells themselves which may overall, negate the 
desired effect on blood platelets, and hence metastasis. 
According to the present invention, the compound 
3-methyl-1-[2-(2-naphthyloxy)-ethyl]-2-pyrazolin-5-one, disclosed and 
claimed as a therapeutically efficacious antithrombotic agent in U.S. Pat. 
No. 4,053,621, has been found to be a potent antimetastatic agent without 
treating the tumor per se. 
SUMMARY OF THE INVENTION 
The present invention is directed to a therapeutic method for reducing 
metastasis in a mammal. The method involves administering to the mammal a 
therapeutically effective amount of 
3-methyl-1-[2-(2-naphthyloxy)-ethyl]-2-pyrazolin-5-one without treating 
the tumor per se.

DETAILED DESCRIPTION OF THE INVENTION 
As disclosed in U.S. Pat. No. 4,053,621, the methyl pyrazolin compound used 
in the present invention (represented by Formula I below), can be prepared 
by various routes of synthesis as illustrated below. According to Process 
A, 2-(2-naphthyloxy)-ethylhydrazine is reacted with an acetoacetic acid 
derivative; according to Process B, 3-methylpyrazolin-5-one is reacted 
with a 2-(2-naphthyloxy)ethyl derivative; according to Process C, 
2-(2-naphthyloxy)-ethylhydrazine is reacted with a tetrolic acid 
derivative. 
##STR1## 
Diluents which can be used include all inert organic solvents, optionally 
diluted with water, e.g., hydrocarbons such as benzene and toluene; 
halohydrocarbons such as methylene chloride; alcohols such as methanol and 
ethanol; and organic basis such as pyridine and picoline. 
Basic or acid condensation agents can be used, and the reaction temperature 
can be varied between 10.degree. and 200.degree. C. The compound can be 
easily purified by conventional means by recrystallization from a suitable 
solvent. 
In the present specification the expression "diluent or carrier" means a 
pharmaceutically acceptable non-toxic substance that when mixed with the 
active ingredient or ingredients renders it suitable for administration. 
The expression preferably excludes water and low-molecular weight organic 
solvents commonly used in chemical synthesis, except in the presence of 
other pharmaceutically necessary ingredients such as salts in correct 
quantities to render the composition isotonic, buffers, surfactants, 
coloring and flavoring agents, and preservatives. Examples of suitable 
solid and liquid diluents and carriers are the following: water containing 
buffering agents which can be rendered isotonic by the addition of glucose 
or salts; non-toxic organic solvents; such as paraffins, vegetable oils; 
alcohols; glycols; natural ground rock (for example kaolins, aluminas, 
talc or chalk); synthetic rock powders (for example highly dispersed 
silica or silicates); and sugars. 
Oral administration can be effected utilizing solid and liquid dosage unit 
forms such as powders, tablets, dragees, capsules, granulates, 
suspensions, solutions and the like. Where appropriate, dosage unit 
formulations for oral administration can be microencapsulated to prolong 
or sustain the release as for example by coating or embedding particulate 
material in polymers, wax or the like. 
Parenteral administration can be effected utilizing liquid dosage unit 
forms such as sterile solutions and suspensions intended for subcutaneous, 
intramuscular or intravenous injection. These are prepared by suspending 
or dissolving a measured amount of the compound in a nontoxic liquid 
vehicle suitable for injection such as an aqueous or oleaginous medium and 
sterilizing the suspension or solution. Stabilizers, preservatives and 
emulsifiers can also be added. 
Generally the parenteral dosage will be from 0.01 to 50 mg/kg, preferably 
from 0.1 to 10 mg/kg, of body weight per day, and the oral dosage form 
will be from 0.1 to 500 mg/kg, preferably 0.5 to 100 mg/kg, of body weight 
per day. 
The following procedure was used to determine the antimetastatic properties 
of 3-methyl-1-[2-(2-naphthyloxy)-ethyl]-2-pyrazolin-5-one without treating 
the tumor per se. The test protocol utilized two unrelated murine tumor 
types (a melanoma and a carcinoma) to minimize the possiblity the the 
results obtained are "unique" to a single tumor type. Both of these tumors 
are routinely used for basic studies on the mechanism of metastasis 
without treating the tumor per se. 
A. IN VIVO MAINTENANCE OF TUMOR LINES 
Subcutaneous B-16 amelanotic melanoma (B-16.sub.a) and Lewis Lung carcinoma 
(3LL) were obtained from the Division of Cancer Treatment, (NCI), Animal 
and Human Tumor Bank, Mason Research Institute, Worcester, Mass. Both 
types of tumors were passaged four times after receipt. Passage involved 
subcutaneous implantation of a 2.times.2 mm tumor dice in the right 
axillary region (using a 13 gauge trocar needle) of male, syngeneic host 
mice [(C57BL/6J; Jackson Laboratory Strain]. The host mice were between 
17-22 g (approximately 28 days old) and housed under identical conditions 
of photoperiod, feeding regimen, temperature, etc. 
The transplanted tumors were allowed to grow in the syngeneic host mice for 
approximately 14 days following implantation. 
B. ISOLATION AND SUSPENSION OF TUMOR CELLS 
Tumor cells were then obtained from the host mice by aseptic removal and 
dispersed using sequential collagenase digestion, as described below. The 
removed tumors were diced (4.times.4 mm) and the diced tissue divided 
(approximately 500 mg/flask) between 6-8 sterile polycarbonate Erhlenmeyer 
flasks. A 10 ml portion of a "tumor dispersion solution" (TDS) was added 
to each flask. 
The TDS was prepared by mixing together Composition A and Composition B 
described below. 
______________________________________ 
COMPOSITION A 
(based on 1 liter) 
______________________________________ 
9.5 g/l Sterile Eagle's Minimum Essential 
Medium (MEM) (Commercially avail- 
able from Gibco, Grand Island, 
New York) 
10 ml/l Nonessential Amino Acids (Gibco) 
10 ml/l Sodium pyruvate 
0.35 g/l Sodium bicarbonate (15 mM) 
5.9 g/l HEPES (25 mM) (an organic buffer; 
commercially available from Sigma 
Chemical, St. Louis, Missouri) 
150 Units/ml Penicillin 
100 .mu.g/ml Neomycin sulfate 
______________________________________ 
The antibiotics were added to ensure that bacterial contamination did not 
occur. 
Composition B is a dry mixture containing collagenase low in clostripain 
and other proteolytic activity; deoxyribonuclease (DNase) to dissolve 
deoxyribonucleoprotein released from damaged cell nuclei; lima-bean or 
soybean trypsin inhibitors to exclude any residual tryptic activity; human 
serum albumin to eliminate non-specific protease activity and absorb 
peroxy and hydroperoxy fatty acids liberated from damaged membranes. 
______________________________________ 
COMPOSITION B 
Weight/ml 
Composition A 
______________________________________ 
Collagenase (Worthington 
1 mg/ml 
type III) 
DNase I (Sigma Chemical) 
50 .mu.g/ml 
Soybean trypsin inhibitor 
100 .mu.g/ml 
(Worthington) 
Human serum albumin (fatty 
10 mg/ml 
acid-free; Sigma Chemical) 
______________________________________ 
Composition B was weighed out and placed in a flask and 100 ml of 
Composition A added. 
The diced tissue in the TDS was then dispersed (30 min., 37.degree. C.) 
under air in a Dubnoff Metabolic Shaker (90 oscillations/minute). 
Supernatants were collected through cheesecloth into sterile 50 ml 
polycarbonate round bottom centrifuge tubes and centrifuged for 10 minutes 
(25.degree. C.) at 900 rpm (100 xg) in a Sorvall SS-34 rotor. Following 
centrifugation, the supernatant fraction was discarded. The solid cellular 
matter (pellets) obtained were washed twice with MEM solution, resuspended 
in MEM and held at 4.degree. C. 
A 10 ml portion of TDS was added to the remaining diced tissue and the 
tissue incubated in a metabolic shaker as described hereinabove, except 
for a period of 60 minutes. The centrifugation was repeated and the 
resuspended cells were combined. 
The cell viability was determined by the vital dye exclusion method [See 
Exptl. Cell Res. 13: 341-347 (1957)]. The cell count was determined in a 
hemocytometer. The stromal cell contamination, e.g. macrophages, red blood 
cells, etc. was determined by visual examination under a microscope. The 
final cell suspension obtained consisted of greater than 99 percent 
monodispersed cells with approximately 25 percent host stromal cell 
contamination. Typical yields from a 3.0 g B-16.sub.a or 3LL tumor ranged 
between 9.times.10.sup.8 and 1.3.times.10.sup.9 viable tumor cells. 
The final cell suspensions were then subjected to centrifugal elutriation 
for final separation of the tumor cells. In centrifugal elutriation, cells 
are subjected to two opposing forces within a separation chamber; a 
centrifugal field generated by a spinning rotor and a counterflow of fluid 
in the opposite (centripatal) direction. A sample suspended in a medium 
enters the separation chamber. Each cell tends to migrate to a zone where 
its sedimentation rate is exactly balanced by the flow rate of the fluid 
through the separation chamber. The chamber's geometry produces a gradient 
of flow rates from one end to the other; cells with a wide range of 
different sedimentation rates can be held in suspension. By increasing the 
flow rate of the elutriating fluid (separation medium) in steps, or by 
decreasing the rotor speed, successive populations of relatively 
homogenous cell sizes can be washed from the chamber. Each population will 
contain cells which are larger or more dense (i.e. faster sedimenting) 
than those of the previous fraction. 
Centrifugal elutriation was accomplished by suspending the tumor cells in a 
"Tumor Resuspension Solution" (TRS), having the following composition, 
based on one liter. 
______________________________________ 
9.5 g/l Sterile Eagle's Minimum Essential 
Medium (MEM) (Gibco) 
10 ml/l Nonessential Amino Acids (Gibco) 
10 ml/l Sodium pyruvate 
0.35 ml/l Sodium bicarbonate (15 mM) 
5.9 g/l HEPES (25 mM) (Sigma Chemical) 
150 Units/ml Penicillin 
100 .mu.g/ml Neomycin sulfate 
______________________________________ 
The suspension was elutriated using a Beckman JE-6 elutriator rotor 
operating at 2000 rpm in a Beckman J-2-21 centrifuge at 25.degree. C. 
A separation medium of Hank's Balanced Salt Solution was pumped through the 
system using a Cole Palmer Master Flex pump with a No. 7014 pump head. The 
pump control box was modified with a 10 turn potentimeter [See Anal. 
Biochem 98: 112-115 (1979)]. The flow rate was measured with a Brooks 
double-ball flow value. 
Hank's Balanced Salt Solution was prepared by preparing a 900 ml solution 
having the following composition and mixing with CaCl.sub.2.2H.sub.2 O as 
described below. 
80 g NaCl 
4 g KCL 
0.98 g MgSO.sub.4 
0.48 g Na.sub.2 HPO.sub.4 
0.60 g KH.sub.2 PO.sub.4 
A 1.85 g portion of CaCl.sub.2.2H.sub.2 O was made up to 100 ml solution, 
and mixed together with the 900 ml described above. 
Approximately 1.times.10.sup.9 cells were injected through an in-line "Y" 
fitting into the mixing chamber. After a 15 minute equilibration time, 
cell debris was eluted at a flow rate of 9.0-10 ml/min. Tumor cells were 
eluted in 6 fractions of 50 ml each at flow rates from about 12-18 ml/min. 
Fractions 2-5 containing tumor cells were combined, recentrifuged 
(100.times.g) and resuspended in 1-2 ml of the TRS described above. 
Recoveries were generally between 70-75 percent of the cells injected into 
the mixing chamber. 
C. EFFECTS OF 3-METHYL-1-[2-(2-NAPHTHYLOXY)-ETHYL]-2-PYRAZOLIN-5-ONE ON 
TUMOR CELL METASTASIS AND GROWTH 
The B-16.sub.a melanoma and Lewis Lung carcinoma cells thus obtained were 
used to test the antimetastatic of the methyl pyrazolin compound without 
treating the tumor per se, as described below. 
Metastasis 
As indicated earlier, metastasis is a single phenomenom represented by an 
intricate series of events. At present, there are two "model" systems 
widely used in studying in vivo metastasis. The first model system 
involves the subcutaneous injection of tumor cells into the animal. 
Subcutaneous injection of tumor cells and subsequent development of a 
primary tumor, followed by spontaneous metastasis is considered to be 
"full" metastasis. Another model system involves the injection of tumor 
cells via the tail vein. Considering the complexity of metastasis, it is 
recognized that tail vein injection is an artificial and only partial 
model system, since it represents events occuring in the latter portion of 
metastasis. However, the tail vein model system is recognized as being 
extremely useful in standardizing experimental conditions. [See Methods in 
Cancer Research, Chapter VII, Academic Press, Inc., 1978.] 
Control (untreated) C57B1/6J mice were tested for full metastasis by the 
following procedure. Cell suspensions of B-16.sub.a and 3LL carcinoma 
cells obtained as described in A and B above, were injected (26 gauge 
needle, 0.2 ml) subcutaneously into the right axillary region of the male 
C57BL/6J mice. Varying amounts of cell suspensions in the range of 
1.times.10.sup.5 to 1.times.10.sup.6 cells were injected. Partial 
metastasis experiments were conducted by injecting the control (untreated) 
mice with tumor cells via the tail vein. The animals were housed under 
identical conditions of temperature, photoperiod, feeding, etc. After an 
observation period of from 17 to 30 days, the animals used in the full 
metastasis and partial metastasis were killed and the lung, liver, kidney, 
spleen and brain tissue was removed. 
The removed tissue was fixed in Bouin's solution. The number of metastatic 
nodules in each organ was determined using a Bausch and Lomb Stereo Zoom 
Microscope. Examination of the control mice for metastatic nodules 
indicated that 100 percent of the animals are positive for metastatic lung 
tumors; the incubation time to produce such metastasis was betwee 17-21 
days and between 23-30 for the 3LL and B-16.sub.a tumor cells, 
respectively. No visible nodules were observed in the liver, kidney, 
spleen or brain tissue. 
The effect of 3-methyl-1-[2-(2-naphthyloxy)-ethyl]-2-pyrazolin-5-one on 
metastasis from tail vein injection of tumor cells and full metastasis 
from subcutaneous injection of tumor cells is shown in Examples 1 and 2 
respectively. 
EXAMPLE 1 
A 3 mg portion of 3-methyl-1-[2-(2-naphthyloxy)ethyl]-2-pyrazolin-5-one was 
suspended in 0.6 ml absolute ethyl alcohol. The suspended methyl pyrazolin 
was dissolved by adjusting the suspension to a pH of 9.5 with NaOH. The 
final concentration of the methyl pyrazolin was achieved by dilution of 
the solution with normal saline (0.9 percent NaCl). 
Syngeneic C57BL/6J host mice were injected on a daily basis, with 0.02 and 
0.08 mg/mouse of the methyl pyrazolin compound (subcutaneously) for a 
period of 3 days. 
On the fourth day, the pretreated mice (and control mice) were injected via 
the tail vein with a 5.times.10.sup.4 B-16.sub.a tumor cell suspension 
prepared as described hereinbefore. The control mice and treated mice were 
housed under identical conditions of temperature, photoperiod, feeding, 
etc. The mice were killed 14 days after tail vein injection of the tumor 
cells and the lung tissue examined. The effect of injecting mice with the 
methyl pyrazolin compound one hour before B-16.sub.a tumor cell injection 
was also determined. 
As seen by the data summarized in Table 1, administration of the methyl 
pyrazolin compound is efficacious in drastically reducing lung tumor 
colonies, i.e., metastasis, at both 0.02 and 0.08 mg levels. 
It has been suggested that the present methyl pyrazolin compound stimulates 
prostacyclin release. [See The Lancet, pp. 518-520 (Mar. 10, 1979)]. The 
antithrombotic activity of prostacyclin is believed to be mediated by 
increasing platelet levels of cyclic adenosine-3',5'-cyclic phosphoric 
acid (cAMP). It is also known that compounds known as phosphodiesterase 
inhibitors slow the breakdown of cAMP. Therefore, by slowing the breakdown 
of cAMP, phosphodiesterase inhibitors would be expected to potentiate the 
anti-thrombotic action of an antithrombotic agent, acting through this 
mechanism. Because platelets may also be involved in the mechanism of 
metastasis, the effect of a well-known phosphodiesterase inhibitor, 
theophylline, was tested for its potential synergism with the methyl 
pyrazolin compound. 
Although the results indicate that the anti-metastatic effect may have been 
enhanced by theophylline, because of the standard error involved in the 
experiment, synergism was not firmly established. 
TABLE 1 
______________________________________ 
Effect of 3-methyl-1-[2-(2-naphthyloxy)-ethyl]-2-pyrazolin-5-one 
on Metastasis from Tail Vein Injected B-16.sub.a 
Amelanotic Melanoma Cells.sup.a 
Lung Tumor 
Treatment Colonies 
______________________________________ 
Control .sup. 181 .+-. 45.sup.b 
0.02 mg 
3-methyl-1-[2-(2-naphthyloxy)- 
19.3 .+-. 7.5 
ethyl]-2-pyrazolin-5-one.sup.c 
0.08 mg 
3-methyl-1-[2-(2-naphthyloxy)- 
2.7 .+-. 1.3 
ethyl]-2-pyrazolin-5-one.sup.c 
Theophylline 200 .mu.g.sup.d 
165 .+-. 38 
Theophylline 200 .mu.g + 0.08 3-methyl-1-[2-(2- 
33.6 .+-. 18 
naphthyloxy)-ethyl]-2-pyrazolin-5-one.sup.d 
0.08 mg 
3-methyl-1-[2-(2-naphthyloxy)- 
65 .+-. 36 
ethyl]-2-pyrazolin-5-one.sup.d 
______________________________________ 
.sup.a 5 .times. 10.sup.4 cells injected intravenously in 50 .mu.l. 
.sup.b -x .+-. SEM; n = 6. 
.sup.c Animals pretreated daily (3 days) before tumor cell injection. 
.sup.d Injected 1 hour prior to tumor cells. 
EXAMPLE 2 
The effect of administration of the methyl pyrazolin compound for an 
extended period of time, on the number of metastatic lung colonies of 
B-16.sub.a and Lewis Lung carcinoma was determined as described below. 
A 3 mg portion of 3-methyl-1-[2-(2-naphthyloxy)ethyl]-2-pyrazolin-5-one was 
dissolved in 0.6 ml ethyl alcohol and the solution adjusted to a pH of 9.5 
with concentrated NaOH. 
Syngeneic C57BL/6J host mice were injected subcutaneously with a 
1.8.times.10.sup.5 B-16.sub.a cell suspension, prepared as described 
hereinbefore. Another series of syngeneic C57BL/6J host mice were injected 
subcutaneously with a 1.times.10.sup.5 Lewis Lung carcinoma cell 
suspension, obtained as described hereinbefore. The day following tumor 
cell injection, the mice were injected subcutaneously for 28 days, with a 
single daily dose of either 0.01 or 0.08 mg of the methyl pyrazolin 
compound. The control mice and the treated mice were housed under 
identical conditions of temperature, photoperiod, feeding, etc. The mice 
injected with B-16.sub.a tumor cells were killed 25 days after injection 
of the tumor cells; the mice injected with Lewis Lung carcinoma cells were 
killed 21 days after injection of the tumor cells. 
Experimental data obtained on the examined lung tissue are summarized in 
Table 2 below. 
TABLE 2 
__________________________________________________________________________ 
Effects of 3-methyl-1-[2-(2-naphthyloxy)-ethyl]-2-pyrazolin-5-one on 
Spontaneous 
Metastasis from Injected B-16.sub.a Amelanotic Melanoma.sup.a and Lewis 
Lung Carcinoma.sup.b 
Lung Tumor Colonies 
Lung Tumor Colonies 
Treatment B-16.sub.a Cells 
Lewis Lung Cells 
__________________________________________________________________________ 
Control 14.1 .+-. 3.1.sup.c (12/12) 
.sup. 34.5 .+-. 6.4 (12/12).sup.c 
0.01 mg 
3-methyl-1-[2-(2-naphthyloxy)- 
1.7 .+-. 0.7 (7/12) 
-- 
ethyl]-2-pyrazolin-5-one 
0.02 mg 
3-methyl-1-[2-(2-naphthyloxy)- 
2.5 .+-. 0.9 (7/12) 
-- 
ethyl]-2-pyrazolin-5-one 
0.04 mg 
3-methyl-1-[2-(2-naphthyloxy)- 
3.1 .+-. 0.8 (8/12) 
-- 
ethyl]-2-pyrazolin-5-one 
0.08 mg 
3-methyl-1-[2-(2-naphthyloxy)- 
1.4 .+-. 0.8 (5/12) 
0.8 .+-. 0.5 (2/12) 
ethyl]-2-pyrazolin-5-one 
__________________________________________________________________________ 
.sup.a 1.8 .times. 10.sup.5 cells injected subcutaneously. 
.sup.b 1 .times. 10.sup.5 cells injected subcutaneously. 
.sup.c Number of metastatic tumor colonies on bilateral lung surface; 
.sup.--X .+-. SEM. 
.sup.d Injected daily subcutaneously in 0.2 ml. 
As shown by the test data summarized in Table 2, the number of metastatic 
lung colonies of both B-16.sub.a melanoma and 3LL carcinoma are 
drastically reduced by administration of the methyl pyrazolin compound. 
With respect to the B-16.sub.a melanoma metastasis, a dosage level of 0.01 
mg appeared to be almost as effective as a dosage level of 0.08 mg. 
As indicated earlier, subcutaneous injection of tumor cells and subsequent 
development of a primary tumor, followed by spontaneous metastasis, is 
considered "full" metastasis. Because the procedure of Example 2 involved 
full metastasis, there were a lesser number of lung tumor colonies present 
in the control animals of Example 2 than in the control animals of Example 
1, which involved development of metastasis from tail vein injection of 
tumor cells. However, the data in both Example 1 and Example 2 indicate 
that the methyl pyrazolin compound possesses strong anti-metastasis 
activity without treating the tumor per se.