NL8201321A - METHOD FOR TREATMENT OF METASTASIS AND GROWTH OF TUMOR CELLS - Google Patents

Description

%, * t APPLICANT SPECIFIES AS INVENTORS: 1. Dr. ir. Wolf-Dieter Busse, 2. Dr. ir. Kenneth V. Honn, 3. Dr. ir. Elke Möller, 4. Dr. ir. Friedel Seuter

Method for the treatment of metastasis and the growth of tumor cells

The primary goal of cancer treatment is the treatment and complete eradication of the primary tumor. Concurrent with this treatment, the prevention of metastasis is necessary, deposits of primary tumor cells and subsequent penetration into the lymphatic system or blood vessels for transmission can be defined. Such extension may occur by adhering to and subsequently penetrating the endothelial walls, depositing secondary tumors in the perivascular tissues, and finally spreading the tumor cells to distant sites.

10 Although much is known about the clinical manifestations of the metastasis process, little is known about the biochemical, immunological, genetic and hormonal mechanisms participating in the metastasis. In this sense, the metastasis can be considered as a separate phenomenon, based on a succession of complicated processes.

Because of the significance of both treatment of primary tumor growth and prevention of metastasis, cancer investigators have conducted extensive studies to elucidate the interactions associated with tumor growth and metastasis.

One of the biological properties that tumor cells appear to possess is the ability to interact with and attach to the platelets of the host, thereby enhancing the ability of the tumor to attach to the microvascular system and to the attach vascular endothelium. As another possibility, it has been proposed that the tumor cells after fixation can dissolve the formation of the platelet-protecting platelets surrounding these cells. In order for a successful metastasis to occur, the metastasis cells must first attach and adhere to the vascular endothelium and remain intravascularly until it penetrates into the surrounding tissue.

___ 3D- Because of the similarities between the process of securing and attaching the tumor cells to the endothelial process and the formation of non-tumor thrombs, many researchers have decided, 8201321 »* ♦ 2, that sites in some way play a role in this. play. Because of this participation, numerous platelets were conducted to determine the effect of anti-coagulant therapy on metastasis. The studies cited below include the administration of anticoagulant compounds, which are potent site aggregation inhibitors. The results so far contradict each other.

Heparin has been reported to both reduce and increase metastasis, especially in the lungs [see Cell Biol.Inti.Rep. 10-2, 81-86 (1963) and Arch. Surg. 91, 625-629 (1965)]. Aspirin gave mixed results [see Eur.J.Cancer J3, (1972) 347-352 and Intl.J.Cancer 11, (1973) 704-718]. Warfarin has been shown to produce significant anti-metastatic effects after intravenous injection of tumor cells and into spontaneously metastatic tumors [see Cancer 35, 15 (1975) 5-14 and Cancer Res. ^ 37, 272-277 (1971)]. It has been shown that metastasis induced by intravenous administration of B-16a-myoma cells can be prevented by administration of the anticoagulant prosta-cyclin [see Cell Biol. 87, (180) 649].

An overview of the application of anticoagulants for tu-20 mor therapy can be found at M.B. Donati et al., Malignancy and the Hemostatic System, pages 103-120, Saven Press, 1981.

It has been suggested that the use of the therapy by means of anticoagulants has therefore in part been less than satisfactory, because the anticoagulants to be used are lacking in specificity and because some of the agents on the tumor cells have self-acting effects. in total, the desired effect on the platelets and thus on the metastasis can be eliminated.

In accordance with the present invention, the compound disclosed in U.S. Pat. No. 4,053,621 and a therapeutically active anti-thrombotic agent is 3-methyl-> 1- [2- (2-naphthyl-oxy) -ethyl] -2- pyrazolin-5-one has been shown to be a potent anti-metastatic agent with accompanying anti-neoplastic activity.

The present invention is directed to a therapeutic method to reduce metastasis and neoplastic growth in mammals. This method involves the administration of a therapeutically effective amount of 3-methyl-1- [2- (2-naphthyloxy) -ethyl] -2-pyrazolin-5-one to the mammals.

As disclosed in U.S. Patent 4,053,621, the methylpyrazolone compound (represented by Formula 1) used in the present invention can be prepared by various routes, as explained below. According to the reaction comparison with Fig. 1, 2- (2-naphthyloxy) ethylhydrazine is reacted with an acetacetic acid derivative; according to the reaction similar to Fig. 2, 3-methylpyrazolinone (3) is reacted with a 2- (2-naphthyloxy) ethyl derivative; 5 according to the reaction comparison with FIG. 3, 2- (2-naphthyloxy) -ethyl-hydrazine is reacted with a tetrolic acid derivative.

Suitable diluents include any inert organic solvents, optionally diluted with water, for example, hydrocarbons such as benzene and toluene, halocarbons such as di-chloromethane, alcohols such as methanol and ethanol, and organic bases such as pyridine and picoline.

Basic or acidic condensing agents can be used and the reaction temperature can be varied between 10 ° C and 200 ° C. The compound can be easily purified by recrystallization from a suitable solvent by conventional methods.

In the present description, the term "pharmaceutically harmless diluent or carrier material" means a non-toxic substance which, after mixing with the active substance or active substances, provides it in a form suitable for administration. The meaning preferably excludes water and conventional low molecular weight organic solvents used in the chemical synthesis, except in the presence of other pharmaceutically required ingredients such as salts in the appropriate amounts, to prepare an isotonic preparation, buffers, surfactants agents, dyes and flavors and preservatives. Examples of suitable solid and liquid diluents and carrier materials are the following: aqueous buffers, which can be made isotonic by adding glucose or salts; non-toxic organic solvents such as paraffin, vegetable oils, alcohols, glycols; milled natural vestiges (for example, cauliflowers, alumina, talc or chalk); synthetic stone powders (e.g. highly dispersed silica or silicates); and sugar.

The oral administration can be carried out using solid and liquid diluents such as powders, tablets, dragees, capsules, granules, suspensions, solutions and the like. Where appropriate, the dosage units for oral administration can be ocapsulated ant to delay or delay over a longer period of time, such as, for example, by coating or embedding particulate matter in polymers, waxes or the like.

Parenteral administration may be in liquid dosage 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 an injectable, non-toxic liquid excipient, such as an aqueous or oil-containing medium, and sterilization of the suspension or solution. Stabilizers, preservatives and emulsifiers can also be added.

In general, the daily weight-related dose for parenteral administration is 0.01 to 50 mg / kg, preferably 0.1 to 10 mg / kg and for oral administration, 0.1 to 500 mg / kg .

The following method was used to determine the antimetastatic and antineoplastic properties of 3-methyl-1- [2- (2-naphthyloxy) -ethyl] -2-pyrazolin-5-one. The test scheme uses two unrelated types of mouse tumors (a melanoma a carcinoma) to eliminate as much as possible the possibility that the results obtained are "unique" with respect to the only tumor type. Both tumors are used as a routine for preliminary studies of the mechanism of metastasis and antineoplastic activity.

A. Obtaining the tumor lines in vivo.

Subcutaneous, amelanotic B-16 melanoma (B-16a) and Lewis lung carcinoma (3LL) were obtained from the Division of Cancer Treatment, (NCI), 25 Animal and Human Tumor Bank, Mason Research Institute, Worcester,

Massachusetts, USA. Both tumor types were subjected to four passages after acquisition. A passage included implanting a 2 x 2 mm tumor block into the right axillary cavity region (using a 0.33 mm * No. 13 Trojan needle) from male, 30 syngenic host mice [(C57BL / 6J; Jackson Laboratory). stamj. The host mice weighed between 17 and 22 g (they were about 28 days old) and were kept under identical conditions with regard to the duration of the exposure to light, the diet, the temperature, etc.

The transplanted tumors were allowed to grow for approximately 14 days after implantation into the syngeneic host mice.

B. Isolation and suspension of tumor cells.

Tumor cells were then obtained by aseptic collection from the host mice and dispersed with subsequent collagenase digestion as described below. The collected tumors were divided into dice (4 x 4 mm) and the diced tissue was divided into 6 to 8 sterile flasks of polycarbonate (about 500 mg / flask). 10 ml aliquot of a "tumor dispersion solution" (TDS) was added to each of the flasks.

The TDS was prepared by mixing together Composition A and Composition B, which are described below.

Composition A (based on 1 1) 10 9.5 g / 1 sterile Eagle's "Minimum Essential Medium" (MEM) (commercially available from Gibco, Grand Island, New York), 10 ml / 1 non-essential amino acids ( Gibco), 10 ml / 1 sodium pyruvate, 0.35 g / 1 sodium hydrogen carbonate (15 ramol), 15 5.9 g / 1 HEPES (25 mmol) a 4- (2-hydroxyethyl) -1-piperazinethane sulfonic acid ( an organic buffer, commercially available from Sigma Chemical, St. Louis, Missouri), 150 units / ml penicillin, 100 µg / ml neomycin sulfate.

The antibiotics were added to ensure that no bacterial contamination occurred.

Composition B is a dry mixture containing collagenase with little clostripalne and other proteolytic activity; deoxyribonuclease (DNase), to dissolve the deoxyribonucleoprotein released from the damaged cell nuclei; lima bean or soy bean trypsin inhibitors, to exclude any tryptic residual action; human serum albumin, to disable nonspecific protease activity and absorb peroxy and hydroperoxy fatty acids released through damaged membranes.

30 Composition B

Weight / ml of composition A.

Collagenase (Worthington Type III) 1 mg DNase I (Sigma Chemical) 50 yg 35 Soybean Trypsin Inhibitor (Worthington) 100 yg

Human serum albumin 10 yg (fatty acid free; Sigma Chemical)

Composition B was weighed and placed in a flask and then 100 ml of the composition A was added.

40 The dice fabric in the TDS was then airborne in an 8201321

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6 Duhnoff metabolic shaker (90 revolutions / min.) Dispersed (30 min. At 37 ° C). The supernatants were collected through a 50 ml polycarbonate round bottom sterile centrifuge tubes on a side cloth and 10 min (25 ° C) at 900 rpm.

5 (100 g) centrifuged in a Sorvall SS-34 Rotor. The above fractions were removed after centrifugation. The resulting cellular material (beads) was washed twice with MEM solution, resuspended in MEM and kept at 4 ° G.

10 ml of TDS was added to the remaining dice tissue and the tissue was incubated in a metabolic shaker as described above, except that the duration was 60 min. Centrifugation was repeated and the resuspended cells were combined.

Cell viability was determined by the vital dye exclusion method [cf. Exptl. Cell Res. 13, (1957) 341-347]. The cell count (cell count) was determined in a Hamocytometer. The flowable cell impurities, for example macrophages, red blood cells, etc. were determined by visual examination under the microscope. The cell suspension finally obtained consisted of more than 99% monodisperse cells with approximately 25% contamination by flowable host cells. Usual 3.0 g yields of a B-16a or 3LL tumor were between 9.

10® and 1.3. 10 ^ viable tumor cells.

The cell suspensions from the final stage were then centrifuged for final separation of the tumor cells. In the centrifugal elutriation, the cells in the interior of a separation chamber were subjected to the action of two opposing forces, namely a centrifugal field generated by a rotating rotor and a counterflow of liquid in the opposite (centripetal) direction. An environmentally suspended sample is introduced into the separation chamber. Each cell tends to go to a zone where its sedimentation rate is leveled exactly by the flow rate of the liquid through the separation chamber. The chamber geometry creates a gradient of flow rates from one end to the other; cells with a wide range of different sedimentation rates can be kept in suspension. By increasing the flow rate of the elutriation fluid (separation medium) in stages or by decreasing the rotational speed, successive populations with relatively homogenous 40 gene cell sizes can be washed out of the chamber. Each population contains 8201321

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7 cells, which are larger or denser (ie faster sedimenting) than those of the. preceding fraction.

The centrifugal elutriation was performed by suspension of the tumor cells in a "tumor resuspension solution" (TRS), which had the following composition based on 1 liter.

9.5 g / 1 sterile Eagle's "Minimum Essential Medium" (MEM) (Gibco), 10 ml / 1 non-essential amino acids (Gibco), 10 ml / 1 sodium pyruvate, 0.35 ml / 1 sodium hydrogen carbonate (15 mmol), 5.9 g / 1 HEPES (25 mmol) (Sigma Chemical), 150 units / ml penicillin, 100 µg / ml neomycin sulfate.

The suspension was eluted using a Beekman JE-6-Elutriator-Rotor, placed in a Beekman J-2-21 centrifuge at 2000 rpm. was used at 25 ° C.

A separation medium from "Hank's Balanced Salt Solution" was used using a Cole Palmer Master Flex pump with a pumphead no.

7014 pumped through the system. The pump control box was centrifuged by a potentiometer with 10 revolutions [see Anal.

Biochem. 98, 112-115 (1979)]. The flow rate was measured using a Brooks double-ball flow meter.

The adjusted Hank saline was prepared by preparing 900 ml of the solution of the following composition and mixing with CaCl 2 · 2 H 2 O as described subsequently.

80 g NaCl 4 g KCl 0.98 g MgSO 4 30 0.48 g Na 2 HPO 4 0.60 g KH 2 PO 4 1.85 g CaCl 2 * 2 ^ 0 were made up to 100 ml solution and mixed with the above 900 ml.

About 1 x 10 4 cells were injected into the mixing chamber through a Y-fitting into the feed system. After 15 minutes before equilibrium adjustment, the cell debris was washed at a flow rate of 9 x 10 ml / min. eluted. The tumor cells were divided into 6 fractions of 50 ml each with flow rates of about 12 to 40 ml / min. eluted. Fractions 2 to 5 containing the tumor cells, 8201321 <8 were pooled, centrifuged again (100 g) and resuspended in 1 to 2 ml of the previously described TRS. Generally, between 70 and 75% of the cells injected into the mixing chamber were recovered.

C. Effect of 3-methyl-1- [2- (2-naphthyloxy) -ethyl] -2-pyrazolin-5-one on growth and metastasis of tumor cells. B-16a melanoma and thus obtained Lewis lung cancer cells were used, as described below, for the investigation of the antimetastatic and antineoplastic activity of the methylpyra-10-zolone compound.

(1) Metastasis

As noted above, metastasis is a separate phenomenon presented by a series of complicated processes. Today, two "model" systems for examining in vivo metastasis find wide application. The first model system involves the subcutaneous injection of the tumor cells into the animal. The subcutaneous injection of the tumor cells and subsequent development of a primary tumor, followed by spontaneous metastasis, is considered "full" metastasis. Another model system involves the injection of the 20 tumor cells through the tail vein. In view of the complexity of the metastasis, the tail vein injection is considered an artificial and only partial model system, as it only shows the processes during the last part of the metastasis. However, the tail vein model system is also considered extremely beneficial in standardizing test conditions [see "Methods in Cancer Research", Chapter VII, Academic Press. Ine., 1978].

As control animals, (untreated) C57Bl / 6J mice were tested for full metastasis according to the following procedure. Cell suspensions of the B-16a and 3LL carcinoma cells obtained as described above under A and B were injected subcutaneously into the right armpit cavity region of the male C57Bl / 6J mice (0.66 mm * no 26 needle; 0.2 ml). Different amounts of the cell suspensions were injected in the range of 1 x 10 ^ to 1 x 10 ^ cells. Partial metastasis tests were performed in such a way that 35 tumor cells were injected into the (untreated) control mice via the tail vein. The animals were kept under identical conditions of temperature, duration of action of light, feeding, etc. After an observation period of 17 to 30 days, the animals used for full metastasis and partial metastasis were sacrificed and the 40 lungs, liver, kidneys, spleen and brain tissue were removed.

8201321 9

The removed tissue was fixed in a Bouin solution. * The number of the metastatic nodes in each organ was determined using a stereo zoom microscope according to Bausch and Lomb. The examination of the control mice for metastatic nodes gave positive results with respect to metastatic lung tumors in 100% of the animals; the incubation period for establishing such metaetases was 17 to 21 days for the 3LL tumor cells and 23 to 30 days for the B-16a tumor cells. No visible nodules were observed in liver, kidneys, spleen or brain tissue.

10 (2) Antineoplastic Efficacy

Antineoplastic activity was measured in vitro by measuring the DNS synthesis of both B-16a and Lewis lung carcinoma cells by a method underlying thymidine incorporation. Cells that synthesize DNS in preparation for cell division are characterized by their ability to take up thymidine. Hence, cell proliferation is linked to DNS synthesis. When the amount of the DNS synthesized by the tumor cells is reduced, this is an indication that cell division and thereby tumor growth is inhibited.

In addition, the antineoplastic activity was determined by measuring growth of the tumor cells in vitro in tissue cultures. This method involves plating or opening a known number of tumor cells to a growth medium and determining the effect of the presence of the test compound on the proliferation of the tumor cells. Finally, the antineoplastic activity was determined in vivo in such a way that tumor cells were injected subcutaneously into the mice and the occurrence of tumor cell growth as well as the weight of the growing tumor cells in untreated control animals and in the pyrazolone compound treated animals were determined.

The action of 3-methyl-1- [2- (2-naphthyloxy) -ethyl] -2-pyrazolin-5-one on metastasis induced by tail vein injection of tumor cells, respectively. on the full metastasis from the subcutaneous injection of tumor cells is shown in Examples I and II.

Example I

3 mg of 3-methyl-1- [2- (2-naphthyloxy) -ethyl] -2-pyrazolin-5-one were suspended in / 0.6 ml of absolute ethanol. The suspended pyrazolone was dissolved by adjusting the pH to 9.5 with NaOH. The final concentration of the pyrazolone was adjusted by diluting the solution 40 with a normal table salt solution (0.9% NaCl).

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Synthetic C57B1 / 6J host mice received a daily injection of 0.02 and 0.08 mg / mouse of the methylpyrazole-5-one compound (subcutaneous) over a 3 day period.

On the fourth day, the pretreated mice (and the control 5 mice) were each injected via tail vein with a suspension of 5 x 10 ^ B-16a tumor cells prepared as described above. The control mice and the treated mice were kept under identical conditions of temperature, duration of action of light, feeding, etc. The mice were sacrificed in the tail veins 14 days after the injection and their lung tissue was examined. The activity of injecting the pyrazolone compound into the mice one hour before the injection of the B-16a tumor cells was also investigated.

As shown in the data compiled in Table A, the administration of the pyrazolone compound is effective in such a way that it drastically reduces the number of the lung tumor colonies, that is, the metastasis, both on the level of 0.08 mg as at that of 0.02 mg.

The present pyrazolone compound is suspected to stimulate the release of prostacycline [see The Lancet, pages 518-520 (March 20, 1979)]. The antithrombotic action of the prostacycline is believed to be mediated by an increase in the level of the cyclic adenosine-3 ', 5'-phosphoric acid (cAMP) in the platelets. It is also known that compounds known as phosphodiesterase inhibitors delay cAMP cleavage. Accordingly, it would be expected that phosphodiesterase inhibitors, by delaying cAMP cleavage, would potentiate the antithrombotic action of an antithrombotic agent acting by this mechanism. Since platelets can also participate in the mechanism of the metastasis, the action of a well-known phosphodiesterase inhibitor, of the theophylline, was investigated for a potential synergy with the pyrazolone compound.

Although the results indicate that the antimastastic action may be enhanced by theophylline, synergism has not been demonstrated with certainty due to the standard error associated with the experiment.

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Example II

The effect of the administration of the pyrazolone compound over a longer period of time on the number of the metastatic lung colonies of the B-16a and the Lewis lung carcinoma was determined as described below »3 mg 3-methyl-1- [2- (2-naphthyloxy) -ethyl] -2-pyrazolin-5-one was dissolved in 0.6 ml of ethanol and the solution was adjusted to pH 9.5 with concentrated sodium hydroxide.

In syngenic C57BL / 6J host mice, a suspension of 10 1.8 x 105 B-16a cells was injected subcutaneously, which suspension was prepared as described above. In another series of syngenic C57BL / 6J host mice, a suspension of 1 x 10 7 Lewis lung carcinoma cells, obtained as described above, was injected subcutaneously. From the day after the injection of the tumor cells, the mice received a subcutaneous injection of a daily unit dose of either 0.01 or 0.08 mg of the pyrazolone compound for 28 days. The control mice and the treated mice were kept under identical conditions of temperature, exposure time of light, feeding, etc. ». The mice that received the injection of the B-16a tumor cells were sacrificed 25 days after the injection of the tumor cells; the mice injected with the Lewis lung carcinoma cells were killed 21 days after the injection of the tumor cells.

The experimental data obtained from the lung tissue examination are summarized in Table B.

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As the test values summarized in Table B show, the number of the metastatic lung colonies, both of the B-16a melanoma and of the 3LL carcinoma, is drastically reduced by the administration of the pyrazolone compound.

In view of the B-16a melanoma metastasis, a dose level of 0.01 mg appeared to be almost as effective as a dose level of 0.08 mg.

As previously outlined, the subcutaneous injection of tumor cells and subsequent development of a primary tumor followed by spontaneous metastasis is considered "full" metastasis. Because the method of Example II involved this full metastasis, the number of lung tumor colonies present in the control animals was smaller than that in the control animals of Example I, where the metastasis developed from the injection of the tumor cells into the tail vein . However, both the data in Example I and the data in Example II show that the pyrazolone compound has a strong antimetase activity.

The antineoplastic activity of the pyrazolone compound was determined by measuring thymidine incorporation as a measure of DNS synthesis.

Example III

B-16a and Lewis lung carcinoma cells were obtained as described above. The dispersed cells were diluted in sterile 25 ml plastic Erlenmeyer flasks to a concentration of 1 x 25 10 cells / ml in MEM. Tritium-labeled Thymidine (½) with a specific activity of 1.85 x 10 "^ to 2.96 x 10" s ~ Vmmol (50-80 Ci / mmol) was added with 7.4 x 10 "^ s ~ V L1 (2 yCi / ml) added to each ml of the cell suspension.

To a series of flasks containing the B-16a and Lewis lung carcinoma cells, the pyrazolone compound was added in amounts of 0.1, 1.0, 10 and 25 µg / ml of the total volume. The flasks were then incubated together with control flasks of the tumor cells, which did not contain pyrazolone compound, at 37 ° C in a Dubnoff metabolic shaker (90 revolutions / min).

At 4 h and 18 h time intervals, 4 equal aliquots up to 1 ml were taken with a pipette and placed into 1.5 ml conical polypropylene tubes. The cells were centrifuged by spinning in a Brinkman mini centrifuge (10,000 g) for 8 minutes. The above fraction was removed and discharged into a radioactive waste reservoir. 1.0 ml of cold trichloroacetic acid 8201321 T * 2 15 acid (6 w / v%) was added to each tube to precipitate the proteins present, including DNS and RNS. The tubes were capped and rotated to break up the cells by centrifugation as described above. The granules obtained contained the acid-insoluble fraction with the DNS. The supernatant containing the acid-soluble ingredients was removed. The inside of each tube was wiped with a cotton swab to remove excess acid soluble radioactivity. The beads were dissolved in each tube by adding 50 µl of tissue dissolution agent, commercially available from Amersham Corporation, Del., Arlington Heights, Illinois, under the tradename NCS. The tubes were capped and incubated at about 50 ° C for about 2 hours or until the granules dissolved.

The tips of the tubes containing the dissolved granules were cut off and the contents were transferred into scintillation ampoules.

3 ml of a 2: 1 scintillation counting solution mixed with xylene were placed in each scintillation vial. Each ampoule was sealed and slung and the amount of the radiolabeled thymidine incorporated by DNS synthesis was determined. The counts per minute were corrected using a Searle PDS computer using the quench correction analysis and indicated as a percentage of the control value.

The test results obtained are shown in fig. Each pyrazolone concentrate involves the amount of the synthesized DNS at the 100% level for the control sample. As fig.

4 shows that the pyrazolone compound effects a concentration dependent decrease in DNS synthesis as determined by the% thymidine incorporation. This decrease in DNS synthesis shows that the pyrazolone compound has antineoplastic activity.

Demonstration of the incorporation of the thymidine in the DNS was performed, such as in Biochem. and Biophysical Research Communications, 35 87, No. 3, pages 795-801 (1979).

Another evidence for the antineoplastic activity of the pyrazolone compound was provided by direct measurement of the proliferation of the tumor cells in tissue cultures.

Example IV

40 B-16a melanoma cells, which were obtained as described above, 8201321 * 16 16, were seeded into 60 mm Petri dishes with grids (gridded) at a density of 3 x 10 4 cells per plate. The cells were cultured in an environment of MEM, Hank basic saline and 10% fetal calf serum, commercially available from Microbiological Associates, Walkersville, Maryland.

After a 24 hour incubation at 37 ° C, the cell counts were performed by visual counting in an inverted microscope. The medium was exchanged and replaced with the above-mentioned medium and pyrazolone in a concentration range ranging from 0.1 to 25 µg / ml. The control groups contain only an exchange of the environment. The pyrazzolone-containing medium was then replaced every second day. After 8 days, cell counts as described above were performed and cell viability was determined by the vital dye exclusive method mentioned above.

The test results obtained are shown in Fig. 5.

The data are presented as mean values of cell number + standard error from parallel tests with six plates. The number above each cross line indicates the viability of the cells, based on a 100 percent viability of the cells. The results show that the pyrazolone compound inhibited the proloperation of the tumor cells over the same dosage range over which the compound also inhibited DNS synthesis.

The viability index, which was in the range between 96 + 0.9 and 94 + 0.4, indicates that the antineoplastic activity of the pyrazolone compound is not due to compound cytotoxicity.

Another assay was performed in vivo to measure the antineoplastic activities of the pyrazolone compound.

Example V

3 mg of 3-methyl-1 ~ [2- (2-naphthyloxy) -ethyl] -2-pyrazolin-5-one were dissolved in 0.6 ml of ethanol and the solution was concentrated with concentrated sodium hydroxide at a pH of 9.5 set.

In syngenic C57BL / 6J host mice, 1 x 10 7 Lewis lung carcinoma cells (0.2 ml) were injected subcutaneously into the axillary cavity region. After a time interval of 24 hours, the pyrazolone compound was administered daily for 21 days. The action of the administration of theophylline together with the pyrazolone compound was also investigated. During the time interval, the mice were fed under identical conditions of temperature, duration of action of light, feeding, etc.

40 held. The mice were sacrificed 22 days after the injection of the tumor cells 8201321 17 and examined by total necropsy for the presence of subcutaneous tumors. Tumors present were removed and weighed to an analysis balance.

The test results obtained are summarized in Table C.

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As the test results in Table C show, both the frequency of occurrence and the weight of the Lewis longearci-noin tuniophen were reduced by the subcutaneous administration of the pyrazolone compound; this demonstrates antineoplastic activity and confirms the in vitro data of Examples III and IV. Theophylline apparently enhanced the activity of the pyrazonlon compound, possibly by increasing the amount of the cAMP.

8201321

Claims (3)

Therapeutic method for the reduction of metastasis and neoplastic growth in mammals, characterized in that a therapeutically effective amount of 3-methyl-1- [2- (2-naphthyloxy) -ethyl] -2-pyrazazolin -5-one or one of its pharmaceutically harmless salts is administered. Method according to claim 1, characterized in that the administration is carried out orally. 3. Process according to claim 1, characterized in that the administration is carried out parenterally. 1-H-1--1 +++ H-8201321