WO1996003996A1

WO1996003996A1 - Deuterium-containing pharmaceutical compositions for destroying tumors

Info

Publication number: WO1996003996A1
Application number: PCT/EP1995/003100
Authority: WO
Inventors: Ulrich Bogdahn; Albrecht Bauer; Axel Haase
Original assignee: Ulrich Bogdahn; Albrecht Bauer; Axel Haase
Priority date: 1994-08-04
Filing date: 1995-08-03
Publication date: 1996-02-15
Also published as: EP0773784A1; AU3257095A; DE4427690A1; CA2196671A1

Abstract

The invention pertains to the use of a pharmaceutical composition containing deuterium and/or deuterated substances and/or substances that enrich or release deuterium to selectively destroy tumor cells and/or tumor metastases or to prevent metastasizing and/or local recurrence of tumors as well as their regrowth.

Description

DEUTERIUM-CONTAINING PHARMACEUTICAL COMPOSITIONS FOR KILLING TUMORS

description

The invention relates to the use of a pharmaceutical composition containing deuterium and / or deuterated substances and / or substances which enrich or release deuterium for the selective killing of tumor cells and / or tumor metastases or for the prevention of metastasis and / or local recurrence of tumors and their regrowth.

The tumor therapeutics and cytostatics developed so far all suffer from the disadvantage that they do not differentiate sufficiently between the tumor cell and the healthy cell. The tumor therapeutics and cytostatic agents developed hitherto also suffer from the disadvantage that they are only insufficiently effective against brain tumors, in particular glioblastomas there, and against melanomas, and do not slow tumor progression sufficiently. This is shown in particular by the mean survival times of less than one year in glioblastoma and melanoma patients. Tumor therapeutic agents against bronchial carcinomas and carcinomas of the gastrointestinal tract are also insufficiently effective.

Previous tumor therapeutic agents also suffer from the disadvantage of causing high side effects even in the lowest concentrations, in particular immune suppression and changes in the blood count and bone marrow depression as well as induction of new tumors.

Some attempts have already been made to treat tumors with D ₂ 0 perform. However, these have so far been unsuccessful or have led to contradictory results. For example, Barbour (Barbour, H. and Allen, E. Tumor growth one fifth saturated with deuterium oxide (heavy water), Am. J. Cancer 32, 1 938) reports on the slower growth of the implanted tumors, but comes to the conclusion that the survival span of the tumor-bearing animals was reduced in a dose-dependent manner by D ₂ 0 application, so that no successful treatment is possible. In particular, high D ₂ 0 concentrations are always classified as toxic.

The latest literature on D ₂ 0 and tumor shows that the withdrawal of D ₂ 0 inhibits tumor growth in the mouse model (Somlyai, G., Jansco, G., Jakli, G., et al.: Naturally occuring deuterium is essential for the normal growth rate of cells, FEBS 31 7, 1 -4 (1 993)), and the presence of D ₂ 0 is a necessary prerequisite for tumor growth.

Previous studies on D ₂ O thus showed that D ₂ O appeared to be of little use as a tumor therapeutic.

The reversibility of the deuterium effects is explicitly mentioned several times in the literature (Gross, P.R. et al., Mitotic Arrest by Deuterium Oxide, Science 1 31, 37-39, 1 960).

The present invention is therefore based on the object of exerting a selective and cytotoxic effect on tumor cells and / or metastases and of largely leaving the growth of normal cells unaffected and of preventing the metastasis and / or recurrence of tumors and / or their regrowth. This would also avoid some of the side effects known for tumor therapeutics.

This object is achieved by the use of a pharmaceutical composition according to patent claims 1, 2, 9 or 10, ie by the use of a pharmaceutical composition containing deuterium and / or deuterated substances and / or substances which induce deuterium. honor or release. Preferred embodiments result from the subclaims.

Systematic studies on the use of deuterium-containing substances as a therapeutic agent against tumors have not yet been carried out, and it has so far been unclear whether differences exist in influencing the growth of tumor cells and normal cells.

Therefore, the applicant has carried out systematic studies on the behavior of tumor cells under the influence of deuterium.

Surprisingly, a direct cytotoxic effect on tumor cells was found. In addition, it was surprisingly found that the direct cytotoxic effect of deuterium is highly selective on tumor cells, i.e. that they are killed to a much greater extent than comparable normal cell populations. Surprisingly, it was also found that the known effects of cell cycle arrest and growth inhibition are likewise highly selective for tumor cells, i.e. that cell cycle arrest and growth inhibition are much more pronounced on tumor cells than on normal cells and thus there is a selective effect on tumor cells.

The direct cytotoxic effect can be demonstrated in vitro on cell culture material. Cells are killed in vitro by adding a deuterium-containing substance or compound to the culture medium. The following% by volume are to be understood as follows. Substance containing 100% by volume of deuterium means that all H atoms at one (or more) specific positions are replaced by D atoms. For example, contains pure D ₂ O, molecular weight 20.03 g / mol, 2 atoms of deuterium (4 g / mol) and one atom of oxygen (1 6 g / mol). If the concentration of a "deuterium-containing substance" is therefore determined, its pure deuterium content in this example is only a maximum of 20% by weight, corresponding to 100% by volume. A suitable concentration for killing over 95% of cells, for example the tumor HTZ-1 9 (malignant melanoma), under suitable culture conditions as a monolayer is over 50% by volume of D ₂ 0 or 99.86 g of deuterium per kg of active substance or another deuterium-containing substance with H atoms replaced by D at a certain point in 50% by volume, preferably over 70% by volume (1 39.81 g deuterium / kg), very preferably over 90% by volume (1 79.75 g deuterium / kg). However, higher or lower concentrations are also suitable for killing tumor cells to a greater or lesser extent.

At a concentration of approx. 10% by volume (1 9.97 g deuterium / kg) D ₂ 0 or deuterium-containing substance, only 5% growth inhibition, but no cell killing, was observed in normal glial cells HTZ-1 40 in the same period .

It has been shown that deuterium has a cytotoxic effect on tumor cells by inhibiting DNA synthesis [ ³ H-thymidine incorporation, Coligan, JE, Kruisbeek, AM, Margulies, DH Shevach, EM, Strober, W., Current Protocols Immunology , NIH Monographie, J. Wiley & Sons, New York, 1 992], and also demonstrated by cell membrane defect [trypan blue staining, GK Smith, Duch, DS, Dev, IK Kaufmann, SH, Metabolie Effects and Kill of Human T-Cell Leukemia by 5-Deaza acyclotetrahydrofolate, a Specific Inhibitor of Glycineamide Ribonucleotide Transformylase, Cancer Res. 52, 4895-4903, (1 992)]. However, the survival of normal, non-degenerate cells is only affected to a lesser extent. Therefore, deuterium-containing substances are suitable for producing a therapeutic agent for killing tumor cells and / or metastases and for preventing metastases and / or the local recurrence of tumors and their regrowth. Such a therapeutic agent is particularly suitable for the therapy of malignant melanomas, gliomas and astrocytomas, bronchial carcinomas (in particular small-cell bronchial carcinomas), neuroblastomas and carcinomas of the gastrointestinal tract. For therapeutic use, deuterium or deuterium-containing or deuterium-releasing or deuterium-enriching substances are usually combined with auxiliaries, fillers and / or additives (for example flavorings, electrolytes, nutrients, vitamins, substances which influence absorption) in one, preferably sterile , isotonic and / or pyrogen-free, pharmaceutical preparation or formulation used, preferably in the form of suppositories, ointments, solutions, dispersions and emulsions, aerosols, foams, particulate agents (for example granules, agglomerates, powder, microbeads and adsorbates), pills , Lozenges, tablets, dragees, capsules or microcapsules, types of chewing gum, tissue, leaf or thread bases, with bandages or bandages, for smoking or inhaling and in carriers, such as liposomes.

For therapy, the administration is preferably local, intracutaneous or transcutaneous; For systemic use, preferably intravenously, by partially exchanging the body water or blood water content (e.g. by dialysis), intraarterially, orally, rectally; for use in cavities, preferably intrapleural, intrathecal, intraventricular, intraperitoneal, intracavitary, in operating theaters. Use in operating theaters is preferably used to prevent relapse of the removed tumor, i.e. to prevent local recurrence and regrowth of the tumor. Extracorporeal killing of tumor cells is also possible (e.g. "purging" in bone marrow neoplasia).

For therapeutic applications, deuterium or deuterium-containing or deuterium-releasing or deuterium-enriching substances are also combined with other active substances, preferably other cytostatics or tumor therapeutics. The newly discovered effect of cell killing and the tumor specificity mean that additive or superadditive (synergistic) effects can be achieved in combination with other cytostatics and tumor therapeutics. This is preferably done by attacking the cytoskeleton, especially by attacking the tubulin. In particular, a reduction in the dose of the combined cytostatic / tumor therapeutic can be expected from a combination with deuterium-containing therapeutic agents according to the invention consequently also a reduction in the side effects, which often restrict therapy.

In particular, a combination with cytostatics / tumor therapeutics is promising, based on the same or similar principles of action as deuterium, namely cell killing (also apoptosis).

Examples of possible combination partners are:

- substances that cause cell death: such as Adriamycin, cisplatin, cyclosporin A, vinca alkaloids

Inhibitory growth factors that can trigger cell death: such as TGF-ß (tumor growth factor ß), TNF (tumor necrosis factor)

Substances that attack the tubulin cytoskeleton, e.g. Vinca alkaloids, taxol, taxol derivatives and colchicine and colchicine derivatives

Alkylating agents: such as Cyclophosphamide, busulfan, ACNU and other nitrous urea derivatives

Purine and pyrimidine antagonists such as e.g. Azathioprine, 6-mercaptopurine, cytarabine

For many cytostatics / tumor therapeutics, the hemotoxic / immunosuppressive (side) effect (reduction in the number of blood cells / in particular lymphocytes and bone marrow depression) is restrictive of therapy. Such an effect is not to be expected from deuterium or deuterium-containing or enriching substances for normal cells, as shown for normal brain cells and in particular lymphocytes (example 6), ie tumor cells are selectively killed. For this reason, the treatment of tumor-infiltrated bone marrow or bone marrow stem cells before bone marrow transplantation ("purging") is used for the selective elimination of tumor cells possible, which can be done intracorporeally as well as extracorporeally. The same effect can therefore be achieved by combining such tumor therapeutic agents with deuterium or deuterium-containing and releasing substances at a lower dose. At the same dose, a greater anti-tumor effect than previously possible can be achieved by combining it with deuterium.

Deuterated tumor therapeutic agents are therefore particularly suitable for combination with

Antimetabolites: such as Methotrexate, thioguanine

Alkaloids: such as Vinblastine, vincristine, taxol

Antibiotics with tumor therapeutic potency: such as Daunorubicin, Bleomycin, Epirubicin

various other bone marrow depressive tumor therapeutics, such as Hydroxyurea, procarbazine and alkylating agents, and purine / pyrimidine derivatives.

A synergistic or potentiating effect also results when the pharmaceutical composition is used in combination with irradiation methods such as gamma radiation, electron radiation (β-radiation), neutrons and corpuscular radiation.

The cell proliferation was determined by means of ³ H-thymidine incorporation, as described for example in "Bogdahn, U., R. Apfel, M. Hahn, M. Gorlach, C. Bohl, J. Hoppe and R. Martin: Autocrine tumor cell growth inhibiting activities from Human Malignant Melanoma, Cancer Res. 49, 5358-5363 (1,989) ". Growth and survival curves are based on cell counting with trypan blue dye.

Solid tumors, such as glioblastoma multiforme, astrocytoma, malignant melanoma, small cell bronchial carcinoma, colon carcinoma, are preferred. Sarcomas, breast cancers, cervical cancers, uterine cancers, prostate cancers, thyroid cancers, pancreatic cancers as well as malignancies of the hematopoietic and lymphopoietic system, treated by the composition according to the invention.

As a control for normal cells, cells from normal brain (after skull trauma), normal brain or IL-2 stimulated (freshly isolated) lymphocytes, PHA stimulated (freshly isolated) lymphocytes can be used.

The effect of deuterium on cells is preferably achieved with D ₂ 0. The suitably concentrated D ₂ 0 was preferably produced in the experiment by diluting high-purity D ₂ 0 (> 99%) with H ₂ 0. D ₂ 0 intended for therapy is preferably produced directly in the desired concentration. However, the effect of deuterium according to the invention can also be achieved by other substances which contain deuterium or which release or enrich deuterium in a suitable manner. This includes all conceivable inorganic or organic compounds that contain deuterium, z. B. especially deuterated amino acids (such as deuterated glutamine, deuterated alanine and deuterated glutamate), deuterated sugars (such as deuterated glucose, deuterated fructose), deuterated fats, deuterated DNA or RNA building blocks (eg deuterated adenine, guanine, cytosine , Thymidine, uracil) and deuterated metabolic products (eg deuterated pyruvate, deuterated lactate) and deuterated metabolic products of the intermediate metabolism.

In summary, it can be stated that the use of D ₂ 0 and other deuterated compounds surprisingly enables successful therapy of tumors and metastases. Metastasis is completely prevented.

In addition to a slowdown in growth, an irreversible destruction of tumors or tumor cells by the effect of "cell kill" is also surprisingly observed. The effect of cell killing is tumor-specific, one The killing of healthy cells is only observed to an insignificant extent.

Contrary to earlier assumptions has D ₂ 0 in a therapeutically effective dose, ie, is observed at a dose at which a -Abtötung of tumor cells, a sufficient compatibility, so that an application over prolonged periods of treatment until complete cure is possible.

The invention will now be further elucidated by the following examples in connection with the attached figures. The pictures show:

Fig. 1, 2, 9, 1 3, 14, 1 9, 23: ³ H-thymidine incorporation in relation to the deuterium concentration,

Fig. 3, 4, 1 0, 1 5, 1 6, 20, 24: growth curves of tumor cells and

Controls,

Fig. 5, 6, 1 1, 1 7, 21, 25: survival rate of tumor cells in relation to the length of stay in media containing deuterium,

Fig. 7, 8, 1 2, 1 8, 22, 26: survival fraction of tumor cells in relation to the length of stay in deuterium-containing media

Fig. 27a + b: Cell cycle of untreated HTZ-1 9 melanoma cells

Fig.28a + b: Cell cycle of HTZ-1 9 melanoma cells after 3 days in 50 vol% D ₂ O

Fig.29a + b: Cell cycle of HTZ-1 9 melanoma cells after 3

Days in 90 vol% D ₂ O Fig. 30: Colony formation rate in soft agar

Fig. 31: Migration area of a tumor

Fig. 32: Comparison of the number of liver metastases in untreated test animals and with D ₂ 0-treated test animals after implantation of a sarcoma M 5076

Fig. 33: Comparison of the number of surviving test animals after intraperitoneal implantation of a sarcoma M 5076

For example:

Determination of the effect of deuterium on tumor lines

To determine the effect of deuterium on tumor cells, exponentially growing HTZ-209B, HTZ 243, HTZ 1 9, HTB 69, HTZ 25, CaCo-2 or HT 29 cells are each used for at least 24 hours in microculture plates with 96 wells (Costar, Zurich) sown in 200 μ \ culture medium with a density of 3000 cells per well [according to Chambard et al. J. Cell. Physiol. 1 35 (1 988), 1 01 -1 07]. The culture medium consists of Dulbecco's MEM with 1 0% FCS addition, 1% vitamins, 1% non-essential amino acids and 0.3% L-glutamine.

A medium change is then carried out and deuterated medium is added. Deuterated media are produced using MEM powder medium and heat-sterilized D ₂ O as well as Ultra Pure Water for dilution with identical additives as conventional culture medium.

The cells are in deuterated medium in the desired concentration / 03996 PC17EP95 / 03100

- 1 1 - incubated for a defined period at 37 ° C, 5% C0 ₂ . After the addition of 1 μCi ³ H-thymidine (specific activity 23 Ci / mol, Amersham Buchler, Braun¬ schweig) per well, the cells are incubated for a further 16 hours and the ³ H-thymidine incorporation into the cellular DNA is then carried out after acid precipitation in the usual way measured using a liquid scintillation counter. The effect of deuterium is expressed as a percentage of the ³ H-thymidine incorporation of the treated cells compared to the ³ H-thymidine incorporation in untreated control cells.

Using different concentrations of D ₂ 0, a concentration can be determined in which the ³ H-thymidine incorporation is inhibited by 50% compared to the untreated control. (IC 50 value).

Tumor IC 50 [% D ₂ 0]

HTZ 209 Glioblastoma multiforme 41

HTZ 243 Astrocytoma, WHO III 9

HTZ 1 9 malignant melanoma 49

HTB 69 malignant melanoma 71

HTZ 25 bronchial carcinoma 39

CaCo-2 colon carcinoma 45

HT 29 colon carcinoma 42

Example 2:

Determination of the cytotoxic activity of deuterium on tumor cells

To determine the cytotoxic effect of deuterium on tumor cells, exponentially growing HTZ-1 9, HTZ 209, HTZ are used analogously to Example 1 243, HTB 69, HTZ 25, CaCo-2 or HT 29 cells sown for at least 24 hours in microculture plates with 96 wells or in microculture plates with 24 wells with a density of 1 0,000 cells per well. As in Example 1, the culture medium consists of Dulbeccos MEM with 10% FCS addition, 1% vitamins, 1% non-essential amino acids and 0.3% L-glutamine. A medium change is then carried out and deuterated medium is added in the desired concentration. The cells are incubated in a concentrated medium in the desired concentration for a defined period of time at 37 ° C., 5% CO ₂ . The cells are then detached with trypsin and trypan blue (Trypan Blue Stain 0.4%, Sigma Chemicals St. Louis) is added in a defined volume. The cell number of vital and damaged cells is counted in the Fuchs-Rosenthal chamber and plotted as a growth curve or survival curve. As a measure of the effect on different tumor cell cultures, the surviving fraction, ie the proportion of surviving cells of the treated group compared to untreated controls, can be given after 3 or 6 days of incubation with 90% by volume of D ₂ O medium.

Tumor SF 3d SF 6d [90 Voi-% D ₂ 0]

HTZ 209 glioblastoma multiforme 0.55 0.07

HTZ 243 astrocytoma, WHO III 0.58 0.32

HTZ 1 9 malignant melanoma 0.25 0.05

HTB 69 malignant melanoma 0.14 0.08

HTZ 25 bronchial carcinoma 0.35 0, 20

CaCo-2 colon cancer 0.08 0.05

HT 29 colon carcinoma 0.04 0.01 Another suitable method for checking the effectiveness of a cytostatic is colony formation in soft agar. After sowing a defined number of cells, the number of colonies formed is counted after 3 weeks and evaluated in relation to the control as "Colony Forming Efficiency" (CFE).

From Fig. 30 it can be seen that the two tested tumors HT-29 (colon carcinoma) and HTZ1 9 (malignant melanoma) show a high effectiveness of D ₂ 0 and below 90 vol% D ₂ 0 there is a decrease by 4 or 5 powers of ten of vital colony-forming tumor cells.

Example 3: Melanoma

Two different tumors, HTZ-1 9 and HTB-69, were tested. The tumors were expanded in MEM with 10 vol .-% FCS addition. ³ H thyrridine assays were carried out in 96-well plates, growth curves in some cases also in 24-well plates.

By means of ³ H-thymidine incorporation (Fig. 1, 2) as a model for the proliferation activity, an inhibition of over 90% with 6-day exposure with 90 vol.% Deuterium (D ₂ O) in the medium could be achieved in both tumors can be detected (HTZ-1 9: 91% HTB-69: 96%).

The growth curves (Fig. 3,4) for HTZ-1 9 showed a significant slowdown in growth at 50% by volume D ₂ 0 (99.86 g deuterium / kg), at 90% by volume D ₂ 0 ( 1 79.75 g D ₂ O / kg) the growth stagnated over the full observation period of 1 3 days. HTB-69 showed a clear absolute decrease in cell count, while the untreated controls showed exponential growth.

The surviving rate (Fig. 5,6), ("surviving rate") represents a measure of the cell death caused by deuterium: after 6 days in 90 vol .-% deuterium dium were found in HTZ-1 9 68% dead cells (sr = 32), in HTB-69 58% dead cells (sr = 42%), after 12 days 78% and 73% dead cells were registered (determined using trypan blue Cell count).

The resulting overall effect from deuterium was measured as a surviving fraction (Fig. 7,8); after 6 days in 90% by volume of deuterium medium, the survival fraction for HTZ-19 was 0.04, for HTB 0.07, after 12 days for both tumors it was less than 0.01. This means a decrease in vital tumor cells under deuterium treatment of more than two powers of ten (2 log-cell kill). The survival fraction of HTB-69 was already less than 0.5 after 1 day.

Experiments were carried out by means of flow cytometry to verify the information obtained from cell counting and growth curves. In addition to information about the vitality of the cells, the use of appropriate dyes provides information about the DNA content of the cell, and thus about its belonging to a certain phase of the cell cycle. For these investigations, Hoechst 33342 was used as a non-specific DNA marker together with ethidium bromide as a vitality marker. Representative of all results from flow cytometry experiments, the results of the melanoma HTZ-19 are to be presented here.

Figures 27a, 28a, 29a show the frequency distributions ("distribution") of HTZ-19 melanoma cells in the cell cycle. A compensation curve (diagram: dashed line) is calculated from the primary data of the frequency of the occurrence of a certain amount of DNA (shown in the diagram as individual measuring points). The distribution functions of the individual cell cycle compartments (diagram: solid lines for G, S and G ₂ phase) are adapted to this compensation curve and the relative frequencies are determined from these distribution functions. This results in the percentage distribution of the cells over the cell cycle compartments, which can be read on the edge of the figures. Figures 27a-29a always show the number of cells (ordinate) depending on their DNA content (abscissa). The ordinate unit is therefore the cell number, the abscissa unit is the relative DNA content in arbitrary units of the fluorescence intensity.

The distribution of the investigated ^* -n HTZ-19 cells resulted in a proportion of 58.1% in untreated cells in d. 3 - phase, 30.3% in the S phase and 11.6% in the G ₂ phase (Fig.27a).

After 3 days of change rr. 50 VD ₂ 0 erc a cell cycle composition with a share of 1% in the ^ hasr 2, 4% in the S-

Phase and 9.5 ^C ,. the G ₂ phase (A 28a). Sor urd _-h the effect of 50 V% D ₂ 0 of the share; er cells ir: G- .se increased, but the proportions of S-Pf.ase and G ₂ -phase cells v *. .linde '.

A 3-day treatment of the same lines with 90% by volume D ₂ O showed a further reduction in the number of cells in the G ₂ phase to only 2.8%, the rest of the cells were in the early S phase and in the G _r phase (Fig.29a). A differentiation between G, -Phase and S-Phase cells is difficult with such a cell cycle and may be artificial. In summary, the cell cycle experiments can certainly be used to conclude that there is a reduction in cells of the G ₂ phase and a dose-dependent accumulation of HTZ-19 melanoma cells in the G phase and / or the early S phase.

By simultaneous application of ethidium bromide and Hoechst 33342 as a double fluorescence and two-dimensional plot of the measured data to obtain a simultaneous statement about • ^"" 'NΛ amount and Vita'ität of cells.

Figures 27b, 28b, 29b show the intensity of the ethidium bromide fluorescence as ordinate ("Y axis") and the intensity of the maximum fluorescence as abscissa ("X axis"). The number of cells is indicated by the blackening and the point density. The intensity for both axes results from the content of fluorescence-stained DNA. In the case of ethidium bromide fluorescence, the intensity is therefore proportional to the DNA content of non-vital cells.

For untreated HTZ-1 9 cells there is a normal cell cycle of the vital cells (Fig. 27b, area 1 or as projection A), only non-vital cells (marked area 2, a total of 1.2%) are found. The distribution of the ethidium bromide fluorescence (B) also shows the vitality of the sample.

A completely different picture emerges after 3 days of treatment with 90% by volume D ₂ O (Fig. 29b): Almost all cells are no longer vital and therefore show ethidium bromide fluorescence.

An evaluation of the survival rate of these two experiments shows a survival rate of 3.8% with 97.8% non-vital cells in D ₂ O compared to 1.2% in the control group.

In contrast, a survival rate of 72.8% was measured with Trypan blue cell counting in HTZ-19 cells after 3 days of treatment, and the effect of D ₂ O was accordingly underestimated.

The cell cycle distribution of the killed cells is striking: almost all cells are in the G phase, a defined G ₂ phase is hardly recognizable. This result shows important additional information for the cell cycle display of the 90 vol% D ₂ 0 treated cells (Fig. 29a): This cell cycle analysis is the distribution of cells that are no longer vital!

After treatment with 50% by volume D ₂ O for three days, there is also a proportion of non-vital cells. Their distribution is again striking: As expected after the cell cycle results (Fig. 28a) and the restriction of killed cells to the G compartment (Fig. 28b), the cell cycle of non-vital cells shows significant numbers only in the G _r phase (and possibly early S phase), a G ₂ peak is hardly noticeable. However, an additional one appears unexpectedly Intensity maximum with somewhat lower fluorescence intensity than in the cells of the G phase (Fig. 28b). This may indicate fragmented DNA.

In summary, there is a very clear potential for deuterium use in melanoma: a high inhibition at 50-90 vol .-% deuterium as an expression for "growth arrest" and a high proportion "cell kill" lead to a significant decline in vital tumor cells .

These investigations were carried out on other tumor cells with similar results.

Example 4: Small Cell Bronchial Carcinoma

Cells from small cell bronchial carcinoma HTZ-25 were tested. The tumors were expanded in MEM with 10 vol% FCS. ³ H thymidine assays and growth curves were carried out in 96-well plates.

The ³ H-thymidine incorporation (Fig. 9) shows an almost linear dose-response relationship which is largely independent of time. A saturation behavior has already occurred on the time scale 1 to 6 days, the effects are evident very quickly in the range of hours. In the case of HTZ-25, a very high effect was achieved with 94% inhibition after exposure to 90% by volume of D ₂ 0 (1 79.75 g of deuterium / kg) in the medium for 6 days.

The growth curves (Fig. 10) show a clear decrease in the treated cells with good growth of the untreated tumor cells.

The survival rate (Fig. 1 1) drops to 80%, with flow cytometry being able to show a cell kill percentage of 56% after 2 days.

The survival fraction (Fig. 1 2) reaches 0, 1 7, and 1 3 after 6 days Days even 0.09, so that the overall effect on the tumor cells at least 90 vol .-% D ₂ 0 concentration represents a full order of magnitude reduction of vital tumor cells.

In summary, in the case of small-cell bronchial carcinoma, it can be stated that an overall good effect is not caused solely by the inhibition of proliferation, but that a notable "cell kill" was also observed.

Example 5: Colon carcinomas

Colon carcinomas of the ATCC cell lines CaCo-2 and HT-29 were tested. The tumors were expanded in MEM with 10 vol% FCS. ³ H thymidine assays and growth curves were carried out in 96-well plates.

The ³ H-thymidine incorporation (Fig. 13, 14) shows an almost linear dose-effect relationship which also tends to be largely independent of time. The effects are evidently also expressed in the range of hours, but the full expression is achieved, in particular in the case of CaCo-2 cells, only after 3 days of incubation. With HT-29 an extremely high effect could be achieved with 98% inhibition after 6 days exposure of 90 vol.% D ₂ 0 (1 79.75 g deuterium / kg) in the medium, CaCo-2 achieved with 94% inhibition similar values after 6 days of incubation.

The growth curves (Fig. 1 5, 1 6) indicate a fulminant absolute decline in the cells in both colon carcinomas, while the untreated controls are in exponential growth.

The survival rate (Fig. 1 7) shows a clear decrease, in particular with CaCo-2, with 39% survival rate, a significant cell kill was achieved after 9 days of treatment. The survival fraction (Fig. 1 8) shows values of less than 0.1 in both colon carcinomas after 3 days (CaCo-2: 0.08, HT-29: 0.04), after 9 days it was in both tumors even below 0.01, ie two powers of ten, reduction of vital tumor cells at 90 vol.% D ₂ 0 concentration.

In summary, it can be stated in colon carcinomas that the "cell kill" has a maximum effect in colon carcinomas.

Example e: Lymphocytes (control; normal cells)

Freshly isolated blood lymphocytes were tested, expanded according to the Ficoll gradient in RPMI medium with 10% by volume of human AB serum addition. The lymphocytes were stimulated with interleukin 2 (IL-2) or phythaemagglutinin (PHA).

In the ³ H thymidine assays (Fig. 19) as a model for the proliferation rate, it was shown for a period of 2-6 days that it was found in all PHA-stimulated lymphocytes when treated with 90% by volume of deuterium medium (1 79.75 g deuterium / kg), inhibition effects occurred. However, even after 6 days of exposure, the inhibition was still below 60%.

IL-2 stimulated lymphocytes showed time-dependent behavior; at 3 and 6 days there was inhibition, a maximum of 65% inhibition was determined at 90% by volume of deuterium.

The growth curves (Fig. 20) show a decrease compared to the controls both with 50 vol.% (99.86 g deuterium / kg) and with 90 vol.% Deuterated medium, but this is significantly less pronounced than with the tested tumors.

The survival rate (Fig. 21) as a measure of the cell death caused by deuterium is significantly reduced only in the IL-2-stimulated cells in 90% by volume of deuterium; there were values up to 60% "survival rate" corresponding to 40 % killed cells measured with 6d IL-2 stimulation.

The survival fraction (Fig. 22) was also reduced only in IL-2 stimulated lymphocytes (up to 0.36 at 6d in 90 vol.% Deuterium medium with II-2 stimulation). The PHA-stimulated cells remain largely unaffected (0 , 8 with 6d PHA stumulation in 90 vol.% Deuterium).

In summary, it can be stated that the system of stimulated lymphocytes lags significantly behind the effects on tumor cells with regard to the inhibition of proliferation and cytotoxicity caused by deuterium. These experiments show that the deuterium-mediated effects are not dependent on proliferation, or at least not all differential effects are only caused by the greater proliferation in tumor cells: the IL-2-stimulated cells with the lower division rate are more strongly inhibited and show higher cell-kill Sensitivity "as the rapidly dividing PHA-stimulated lymphocytes, which are stimulated even in growth by low doses of deuterium and have hardly any" cell kill ".

Example 7: Gliomas

Cells of the glioblastoma (glioblastoma multiforme) HTZ-209 and the astrocytoma HTZ-243 (astrocytoma WHO grade III) were tested. The tumors were expanded in MEM with 10 vol% FCS. ³ H thymidine assays were carried out in 96-well plates, growth curves were carried out in 24 and 96-well plates.

The ³ H-thymidine incorporation (Fig. 23) shows a clear shoulder formation in the dose range between 1 and 50 vol.% D ₂ O, which is largely independent of time. Here a very clear tumor selectivity can be seen, in particular when using D ₂ 0 over 6 days, with HTZ-243 having already achieved 10% D ₂ 0 over 60% inhibition. The growth curves (Fig. 24) indicate an absolute decline in the cells in both gliomas, especially HTZ-209, while the untreated controls are in exponential growth.

The survival rate (Fig. 25) shows a clear decrease in both tumors, especially in HTZ-209, with 33% survival rate, a high degree of tumor cell kill was achieved after 6 days of treatment.

The survival fraction (Fig. 26) shows an almost linear decline in both brain tumors, and after 6 days for HTZ-209 values below 0, 10, i.e. a full power of ten decrease in vital tumor cells.

In summary, it can be stated in the brain tumors examined that therapeutic success can be achieved by killing tumor cells.

It could be clearly demonstrated by Examples 1-7 that there are two completely different mechanisms for the action of deuterium on cells: the effect of the growth arrest and the effect of the first discovered by the inventors direct cell kill ("cell kill").

The specificity of the effects on certain cell groups has not been systematically examined in the literature. Here too, indications of tumor-specific behavior could be determined for the first time.

The lymphocytes, in which both "growth arrest" and "cell kill" are significantly less pronounced in the more rapidly proliferating PHA-stimulated cells, indicate that the extent of the effects is not only dependent on the proliferation rate. / 03996 PC17EP95 / 03100

- 22 - Example 8: Prevention of metastasis

A suitable method for checking the effect of a cytostatic on the ability to migrate and thus the metastasis of a tumor is to assess the migration of tumor spheroids. Tumor cells are converted back into uncoated plates after the formation of spheroids on agricultured 48-well plates. The tumor cells return to adherent growth within 72 hours; the area covered is a measure of the migration and invasion behavior of a tumor and thus of its ability to metastasize and its inhibition.

In Fig. 31 it can be seen that D ₂ 0 leads to a reduction in the migration area to 20% of the otherwise occupied area after 72 hours, which corresponds to a significant reduction in the ability to metastasize or to form metastases. The cells migrated to this area, which was reduced to 20%, are also devitalized (demonstrated by trypan blue staining), since D ₂ 0, in particular, completely kills off the migrating cells. The use of D ₂ O therefore prevents potential metastasis.

In another experiment, sarcoma M 5076 was implanted subcutaneously in mice. Half of these mice were treated with 50% by volume D ₂ O.

From Fig. 32 it can be seen that the animals treated with D ₂ O did not develop liver metastases, while the untreated control animals got up to 500 liver metastases. The controls showed an average of 1 9.5 liver metastases. The use of D ₂ O thus completely prevents metastasis.

Example 9: Checking the effectiveness in the animal model

1 6 mice were given 10 ⁵ cells of sarcoma M 5076 intraperitoneally. Half of the mice (8 animals) became 50% by volume D ₂ 0, the other / 03996 PC17EP95 / 031 0

- 23 - half (8 animals) given normal water as drinking water (control).

From Fig. 33 we see that 30 days after the Im¬ of the eight control animals plantation only two survived, while another 7 were from the D ₂ 0-treated animals alive. Treatment was discontinued after 35 days. That day only 1 control mouse was alive, but still 7 D ₂ 0-treated mice. The 6 animals from the D ₂ 0 group still alive at the end of the experiment can be considered cured for the purposes of the experiment.

Claims

claims

1 . Use of a pharmaceutical composition containing deuterium and / or deuterated substances and / or substances which

Enrich or release deuterium for the selective killing of tumor cells and / or tumor metastases.

2. Use of a pharmaceutical composition containing deuterium and / or deuterated substances and / or substances that

Enrich or release deuterium to prevent metastasis and / or local recurrence of tumors and their regrowth.

3. Use according to claim 1 or 2, characterized in that the pharmaceutical composition for killing degenerate cells in concentrations above 50 vol .-% (99.86 g deuterium / kg) D ₂ 0 or another deuterium-containing substance with 50 % is replaced by D replaced H atoms.

4. Use according to any one of claims 1 -3, characterized in that the pharmaceutical composition in the form of suppositories, ointments, solutions, dispersions or emulsions, aerosols, foams, tablets, tablets, pills, lozenges tablets, dragees, capsules or Microcapsules, types of chewing gum, tissue, sheet or thread bases in bandages or bandages, for smoking or inhalation and in carriers are used.

5. Use according to one of claims 1-4, characterized in that the pharmaceutical composition for therapy locally, intracutaneously, transcutaneously; for systemic use intravenously, by partially exchanging the body water or blood water content, intraarterially, orally, rectally; for use in cavities, intrapleural, intrathecal, intraventricular, intraperitoneal, intracavitary, administered in operating theaters and used for the extracorporeal killing of tumor cells.

6. Use according to one of claims 1-5 for the therapy of malignant melanomas, gliomas, astrocytomas, bronchial carcinomas, neuroblastomas, gastrointestinal carcinomas, sarcomas, malignancies of the hematopoietic and lymphopoietic system, breast carcinomas, cervical carcinomas, uterine carcinomas , Prostate cancer, thyroid cancer and pancreatic cancer.

7. Use according to any one of claims 1-6, characterized in that the pharmaceutical composition additionally contains cytostatics or tumor therapeutics.

8. Use according to any one of claims 1-7 in combination with irradiation methods.

9. Use of deuterium and / or deuterated substances and / or substances which enrich or release deuterium for the production of a pharmaceutical composition for the selective killing of tumor cells and / or tumor metastases.

10. Use of deuterium and / or deuterated substances and / or substances which enrich or release deuterium for the production of a pharmaceutical composition for preventing meestasis and / or local recurrence of tumors and their regrowth.

1 1. Use according to claim 9 or 10, characterized in that the pharmaceutical composition for killing degenerate cells in concentrations above 50% by volume (99.86 g deuterium / kg) D ₂ 0 or another deuterium-containing substance with 50% D replaced H atoms is applied.

1 2. Use according to any one of claims 9-1 1, characterized in that the pharmaceutical composition in the form of suppositories, ointments, solutions, dispersions or emulsions, aerosols,

Foams, particulate agents, pills, lozenges, tablets, dragees, capsules or microcapsules, types of chewing gum, tissue, sheet or thread bases in bandages or bandages, for smoking or inhalation and in carriers are used.

1 3. Use according to any one of claims 9-1 2, characterized in that the pharmaceutical composition for therapy locally, intracutaneously, transcutaneously; for systemic use intravenously, by partially exchanging the body water or blood water content, intraarterially, orally, rectally; for use in cavities, intrapleural, intrathecal, intraventricular, intraperitoneal, intracavitary, administered in operating theaters and used for the extracorporeal killing of tumor cells.

1 4. Use according to any one of claims 9-1 3 for the therapy of malignant melanoma, glioma, astrocytoma, bronchial carcinoma, neuroblastoma and gastrointestinal carcinoma, sarcoma, malignancy of the hematopoietic and lymphopoietic system, breast cancer, cervical cancer, uterine cancer , Prostate cancer, thyroid cancer and pancreatic cancer.

1 5. Use according to any one of claims 9-14, characterized in that the pharmaceutical composition additionally contains cytostatics or tumor therapeutics.

1 6. Use according to any one of claims 9- 1 5 in combination with irradiation methods.