JPH0643303B2 - Pharmaceutical composition having anticancer activity and anticancer activity enhancer - Google Patents

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition having activity as an anticancer agent and an anticancer activity enhancer.

[Conventional technology and problems]

It has been known for some time that levels of ascorbic acid (vitamin C) are often low in cancer patients, which may be due to increased utilization of ascorbic acid by the tumor cells. Therefore, it was suggested that ascorbic acid could be a useful anti-cancer therapeutic agent in anti-cancer treatment. As reviewed by Cameron et al. (Cancer Res., 39: 663-681, 1979).
Years), several clinical trials have shown that the survival time of cancer patients receiving ascorbic acid is significantly increased. However, ascorbic acid is not widely used as an anticancer drug today.

Recently, it has been proposed to form an adduct of ascorbic acid with other compounds to provide a novel anticancer drug. In this way, for example, Elven
(Elvin) et al. Reported that ascorbic acid adducts with aldehydes such as methylglyoxal and acetylacrolein inhibit the growth of Ehrlich ascites tumor in mice (Eur.J.Cancer Clin.Oncol, 17 ( 7)
Vol., 1981, pp. 759-65). Furthermore, the use of ascorbic acid, in particular ether, and its ketals and acetals as angiogenesis inhibitors has been proposed in EP-A-00086544. Angiogenesis refers to the method of growth of new blood vessels, eg, new blood vessels involved in tumor growth.

Furthermore, recently, 5,6-O-benzylidene-L
-It has been disclosed in EP-A-0148094 that ascorbic acid and its metal salts exhibit anti-cancer properties. The compound can be prepared by reacting L-ascorbic acid with benzaldehyde or, for example, α, α-dimethoxytoluene in the presence of zinc chloride, and has the chemical structural formula shown below.

[Means for Solving the Problems] At present, the cytotoxic effect of 5,6-O-benzylidene-L-ascorbic acid and its metal salts on human cancer cells has been reported to be the reason why if the present anticancer agent is L-ascorbic acid or It was accidentally found to be synergistically increased if co-administered with salt. The ability of 5,6-O-benzylidene-L-ascorbic acid and its salts to enhance the anti-cancer effects of L-ascorbic acid opens up the possibility of new approaches to the treatment of cancer patients. Become.

We also found that at least a similar synergistic effect was obtained by deuterated 5,6-O at the 1-position of the aldehyde group of the benzylidene moiety.
-Benzylidene-L-ascorbic acid and has also been shown to be present in pharmaceutically acceptable salts thereof.

Thus, the present invention provides pharmaceutical compositions useful as anti-cancer agents. The pharmaceutical composition as an active ingredient,
(I) L-ascorbic acid or a pharmaceutically acceptable salt thereof, and (ii) 5,6-O-benzylidene-L-ascorbic acid, deuterated at the 1-position of the aldehyde group of the benzylidene moiety 5,6 -O-benzylidene-L-ascorbic acid and pharmaceutically acceptable compounds selected from the salts of said acids.

The present invention provides 5,6-O-benzylidene-L containing L-ascorbic acid or a pharmaceutically acceptable salt thereof.
An anticancer activity enhancer for a compound selected from ascorbic acid, 5,6-O-benzylidene-L-ascorbic acid deuterated at least at the 1-position of the aldehyde group of the benzylidene moiety and a pharmaceutically acceptable salt; It also includes.

The deuterated 5,6-O-benzylidene-L-ascorbic acid, which is of benefit here, may be deuterated in other parts of the molecule, as well as partially or completely. That is, in addition to the deuterated atom at the 1-position of the aldehyde group of the benzylidene moiety, one or more hydrogen atoms may be replaced by deuterium atoms in the structure.

〔Example〕

The anti-cancer synergistic effect mentioned in the present invention is demonstrated by the experiments described below. In these experiments, we used L-ascorbic acid (abbreviated Asc) and 5,6-O.
-Benzylidene-L-ascorbic acid sodium salt (abbreviated as BASS) or 5,6-O-benzylidene-L deuterated at the 1-position of the aldehyde group of the benzylidene moiety
-The sodium salt of ascorbic acid (abbreviated as BASS-d ₁ ) was used.

Example 1 Biological Materials and Methods Used to Demonstrate Synergistic Effect Cell Culture Technology: Established system NHIK3025 (Nordbye,
K., and Oftebro, R. Exp. Cell Res., Vol. 58, p. 458, 1
969; Oftebro, R., and Nordbye, K., Exp. Cell Res., 5th.
8: 459-460, 1969) is a culture medium E2a (Puck, TT et al., J. Exp. Med., Vol.
-165, 1957) 20% human serum and 10
% Horse serum (Grand Island Biological Co.) was supplemented and cultured. Cells grow as monolayers in tissue culture flasks. After attachment, cells do not move to the surrounding area, and for several generations of cells, inverted microscopes have been used.
The quality is retained so that the same cells are observed at. The cells are maintained in continuous exponential growth by frequent re-culturing, that is, by repeating the culture every second and third days.

Cell viability: To measure cell viability, an appropriate number of cells were seeded in a plastic Petri dish (φ = 5 cm). The number of cells seeded in each dish was adjusted so that the number of viable cells was about 150 per dish. On the other hand, exponentially growing (asynchronous) cells were trypsin digested before seeding, and synchronized cells were seeded immediately after selection. After about 2 hours, the cells were attached to the bottom of the dish and the treatment was started by replacing the culture medium with a medium containing an appropriate drug concentration. After treatment for the desired time, the drug-containing medium was removed and fresh medium was added. The dishes were washed once with the same culture medium added when adding or removing the drug. After 10 to 12 days at 37 ° C in a CO ₂ incubator,
Cells were fixed with ethanol and stained with methylene blue before colonies were counted.

Protein synthesis: The rate of protein synthesis is calculated by learning (Rφnning, φW)
Calculated as described by J. Cell Physiol., 10th.
7: 47-57, 1981). Briefly, the cellular protein is ( ¹⁴ C) valine with a certain specific activity (0.5 Ci / mol) and is pre-incubated for 2 days prior to the experiment and labeled to saturation. It was This was achieved by using high concentrations of valine so that the dilution of ( ¹⁴ C) valine by intracellular valine or valine produced by proteolysis was negligible (Rφnning,
φ. W., et al., Exp. Cell Res. 123, 63-72, 1
979). In this way the specific activity was kept at a constant level. The rate of protein synthesis was calculated from the uptake of ( ³ H) valine with constant specific activity. Uptake was determined by measuring ( ¹⁴ C) protein at the beginning of each measurement period.
It relates to the total radioactivity and is expressed as a percentage per hour (Rφnning, φW, et al., J. Cell, Physiol.
7: 47-57, 1981).

Biological effects The cytotoxic effects of BASS and ascorbic acid (Asc), either alone or in combination, were:
Asynchronous exponentially growing NHIK3025 cells were examined. Cells were treated at 37 ° C for 24 hours and attached to the bottom of plastic Petri dishes. The number of cells that were able to form colonies after treatment was recorded. The results of one experiment are shown in Table 1. Active ingredients (ie OmM BASS and Om
The viability of cells treated with culture medium without M Asc) was defined as 1.0 and was used as a control.

The data in Table 1 demonstrate that the combined use of BASS and Asc induces a stronger cytotoxic effect than BASS or Asc alone, respectively. For example, using 1.0 mM BASS alone, viability is 0.76. Similarly, with 1.2 mM Asc alone, the survival rate is 0.8. Thus, if the two drugs used in combination induced only additional mutually independent effects, the survival rate was 0.
It will be 76 x 0.8 = 0.61. However, from Table 1, the survival rate after treating the two drugs in combination at the above concentrations is 0.16. That is, the combined treatment produces a synergistic effect rather than just an additional effect, resulting in an approximately 4-fold increase in cytotoxicity.

Example 2 An experiment similar to that reported in Table 1 above was further conducted to obtain the data summarized in Table 2.

From Table 2, the same synergistic effect of the present invention can be seen again.

Example 3 As shown in Table 3 below, the same synergistic effect is demonstrated even in a short-time treatment, for example, 4 hours.

Example 4 Our unpublished pending UK patent application No. 87057.
In No. 80, we have shown that certain aromatic aldehydes and their derivatives have an effect of enhancing the anticancer effect in vivo when at least in the 1-position of the aldehyde group.
Therefore, we conducted an experiment to test the cytotoxic effect of deuterated BASS (BASS-d1) when used with ascorbic acid. The results are shown in Table 4 below.

From Table 4, it seems that the synergistic effect of BASS-d1 is at least somewhat high at high Asc concentrations,
Alternatively, it can be seen that the synergistic effect when BASS-d1 is used is demonstrated at an almost equivalent level.

Example 5 To determine if a synergistic effect is obtained with other sugars,
We use BASS in combination with glucose for 24 hours NHIK
3025 cells were processed. The experimental results are shown in Table 5 below. In the table, cell viability is recorded after treatment with 1.6 mM BASS in combination with various concentrations of glucose added to the cell culture.

From Table 5, it can be seen that glucose does not improve the cell-inactivating ability of BASS in any way. And there is no statistically significant improvement in the cytotoxicity of BASS both in the absence of glucose and in the presence of glucose up to 8 mM.

We also studied the effect of using L-ascorbic acid in combination with other aromatic aldehydes or aldehyde derivatives such as 4,6-O-benzylidene glucose, but we can observe any synergistic effect. I couldn't do it.

Example 6 It is known from early studies that the determination of protein synthesis inhibition is a valuable indicator of anti-cancer activity. In the present study, we tested protein synthesis inhibition induced by BASS and Asc alone and induced by the combination of two active agents simultaneously. This experiment also included a similarly treated sample. However, BASS-d1 was used here instead of BASS. The results are shown in Table 6 below.

From the results shown in Table 6, it can be seen that the rate of protein synthesis in the control is 4.5% / hour (average percentage of total cellular protein per hour). BASS or Asc
The use of either of them alone at 3.2 mM resulted in a decrease in the rate of protein synthesis by 1.5-1.7% / hour. However, when both drugs are used simultaneously, 1.
Concentrations of 0 mM BASS and 1.0 mM Asc are sufficient to reduce the rate of protein synthesis to this level (1.73% / hr).

From the data given in Tables 1-6 above, one compound
It is clear that BASS or BASS-d1 and the compound Asc on the other hand, when used together do not act independently. Moreover, the synergistic effect of Asc and BASS or BASS-d1 is stronger than the effect of using one drug alone at the same concentration as the total concentration when used in combination. This indicates that the synergistic effect induced by the two drugs BASS and Asc must result from a reaction product between the two or some cellular sensitization. This sensitization may be the result of increased uptake into cells, inhibition of repair processes, or simply decreased chemical recovery. However, at this time, the explanation for our observed synergistic effect is unknown.

Although the above experiments used the sodium salt of 5,6-O-benzylidene-L-ascorbic acid and the partially deuterated derivative, other pharmaceutically acceptable salts, especially other pharmaceutically acceptable salts, were used. It is also within the scope of the present invention to use possible alkali and alkaline earth metal salts. However, sodium salts are preferred because they are well soluble in water.

Similarly, in some cases, it may be preferable to use a pharmaceutically acceptable salt of L-ascorbic acid, rather than the free acid itself.

In addition, it has been confirmed that the pharmaceutical composition of the present invention exhibits anticancer activity in vivo in mice.

Pharmaceutical compositions containing the active ingredients of the invention may be formulated with pharmaceutically acceptable excipients or carriers by a number of methods well known to those of ordinary skill in the art. The pharmaceutical composition according to the present invention may be administered orally or parenterally. For oral administration, the composition is in the form of tablets, capsules, granular dragees or powders. For parenteral administration, the composition can be in a form suitable for injection or intravenous drip, or can be used as a suppository.

The active ingredient of the composition can be formulated by encapsulation with conventional pharmaceutically acceptable carriers which are either solid or liquid. For example, surface active agents, vehicles, lubricants, adjuvants and pharmacologically acceptable film forming materials can be used as is well known in the pharmaceutical formulation art.

The content of the active ingredient in the pharmaceutical composition according to the present invention can be changed depending on the form of the preparation. When administered orally, 5,6-
The content of O-benzylidene-L-ascorbic acid or pharmaceutically acceptable salt is generally about 0.1 to 20% by weight and is included in the composition together with an equivalent amount of ascorbic acid or the salt. For parenteral administration, the amount of each active ingredient is about 0.01 in order to be absorbed through the mucous membrane.
-10% by weight, the preferred ratio would be 1: 1. Appropriate amounts will be applied to the deuterated compound.

Although it is preferred to formulate the two active ingredients together in a pharmaceutical composition to ensure that the patient receives the desired dose of each active ingredient in order to obtain the optimal therapeutic effect,
If the patient receives the correct dose of each active ingredient for which a synergistic effect is apparent, it is within the scope of the invention to administer each active ingredient separately.

Suitable dose ranges for each of the active ingredients are as follows:
For oral administration, 5,6-O-benzylidene-L-
Ascorbic acid or its salt or deuterated aldehyde derivative may be used in an amount of 10 to 75 per 1 kg of body weight up to twice daily.
It is in the mg range. The acceptable range for ascorbic acid or its salt is 10 times per kg body weight up to twice daily.
Will be ~ 100 mg. For intravenous infusion or injection, a suitable range for 5,6-O-benzylidene-L-ascorbic acid or its alkaline earth salts or deuterated compounds is 10 per kg body weight up to twice a day.
Will be ~ 75 mg. Ascorbic acid buffered with sodium ascorbate or sodium carbonate,
It may be administered at physiologically acceptable levels, usually up to twice daily, at about 10-100 mg / kg body weight in combination with the drug.

Thus, for adult patients, 1.5-3g BASS
Alternatively, it is preferable that BASS-d1 is combined with 5 g or less of L-ascorbic acid or a salt thereof and orally administered per day. For infusion, an adult patient is 1.5
Preferably ~ 3g of BASS or BASS-d1 is administered in combination with 5-10g of either L-ascorbic acid or salt thereof, or alone. In the case of the latter alone, L-ascorbic acid or the salt thereof,
It is then also preferable to administer alone.

Physiological in case of drug combination by injection or drip
The pH will be established by including buffers, as is known in the pharmaceutical industry.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Erik Olai Pettersen Norway, 0594 Oslo 5, Stadtslad Matisse Vey 15 (72) Inventor John Michael Dornish Norway, 0767 Oslo 7, Landing Sveien 136

Claims (8)

[Claims] 1. (i) L-ascorbic acid or a pharmaceutically acceptable salt thereof, and (ii) 5,6-O-benzylidene-L-ascorbic acid, at least a heavy salt at the 1-position of the aldehyde group of the benzylidene moiety. A pharmaceutical composition for treating cancer, which comprises as an active ingredient a compound selected from hydrogenated 5,6-O-benzylidene-L-ascorbic acid and a pharmaceutically acceptable salt of the above acid. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt is selected from alkali metal and alkaline earth metal salts. 3. A pharmaceutical composition according to claim 1 or 2, wherein the active ingredient (ii) comprises a compound of the formula:
[In the formula, X represents hydrogen or a pharmaceutically acceptable metal cation] 4. The pharmaceutical composition according to any one of claims 1 to 3, which comprises a pharmaceutically acceptable carrier or excipient for oral administration. 5. The pharmaceutical composition according to claim 4, which comprises 0.1 to 20% by weight of the active ingredient (i) in an amount equal to that of the active ingredient (ii). 6. The pharmaceutical composition according to any one of claims 1 to 3, which comprises a pharmaceutically acceptable carrier or excipient for parenteral administration. 7. The active ingredients (i) and (ii) are each added in an amount of 0.1 to 0.1.
The pharmaceutical composition according to claim 6, comprising 10% by weight. 8. 5,6-O-Benzylidene-L having L-ascorbic acid or a pharmaceutically acceptable salt thereof.
An anticancer activity enhancer of a compound selected from ascorbic acid, 5,6-O-benzylidene-L-ascorbic acid deuterated at least at the 1-position of the aldehyde group of the benzylidene moiety, and a pharmaceutically acceptable salt.