GB2036554A - Anthraquinones for use in therapy - Google Patents

Abstract

The anthraquinones of formula (I> <IMAGE> wherein R is an alkylene group having 1 to 4 carbon atoms and preferably is an ethylene group, and pharmaceutically acceptable salts thereof preferably the acetate or diacetate, are useful in treatment of the human body for therapy of cancer, especially of leukaemia.

Description

SPECIFICATION Anthraquinones for use in therapy This invention relates to a group of substances for use in a method of treatment of the human body by therapy, namely to substances for use in cancer therapy. The substances to which this invention relates are valuable in particular for therapy of leukaemia.

The substances of this invention are a class of anthraquinones, the preferred anthraquinone being per sea known compound which is part of the state of the art and is disclosed, as a dyestuff, in U.S. Patent 2,051,004, together with the pharmaceutically acceptable salts thereof. The preferred salt is the acetate or diacetate.

The anthraquinones of this invention are those of the general formula

wherein R is an alkylene group of 1 to 4 carbon atoms and the preferred (and known) anthraquinone is that of formula (I) wherein R is ethylene.

These anthraquinones and their salts, the substances of this invention, may be compounded with pharmaceutically acceptable carrier to form a composition for administration to a patient. Such compositions are the subject of our co-pending Application No. 27474/78 (Serial No. 2000029).

We have found thatthe preferred compound of this invention shows presumptive activity against leukaemia in mice, as standard test animals.

Each of the compounds of formula (I), and their pharmaceutically acceptable salts, can be expected to show activity against a broad range of cancer diseases, and especially blood cancer disease such as leukaemia, in humans at doses below toxic levels.

The anthraquinone compounds of formula (I) may be prepared as described in Example 59 of United States Patent No. 2,051,005, or by reacting 2 moles of an N - aminoethyl - alkanolamine with 1 mole of leuco quinizarine and oxidising the resulting product with, e.g. air.

The N-aminoalkylalkanolamines useful in preparing the anthraquinones of formula (I) are: N - aminomethylmethanolamine (H2NCH2NHCH20H), N - aminoethylethanolamine (H2NCH2CH2NHCH2CH2OH), N - aminopropylpropanolamine (H2NCH2CH2CH2NHCH2CH2CH2OH) and N - aminobutylbutanolamine (H2 NCH2CH2CH2CH2NHCH2CH2CH2CH2OH).

While the reaction mechanism requires 2 moles of N - aminoalkylalkanolamine per mole of leuco quinizarine, it is generally preferred to employ an excess, e.g., 2.5 to 3 moles of the N - aminoalkylalkanolamine to ensure formation of the desired product.

Conveniently, these anthraquinones can be prepared by dissolving the leuco quinizarine in a solvent, such as ethyl alcohol, charging the N aminoalkylalkanolamine with agitation and heating to about 75-80"C. for a time sufficient to allow the reaction to occur. Thereafter, oxygen is bubbled through the solution until the leuco product is oxidised. The solution is cooled to about 10"C., filtered, washed with alcohol and dried.

The pharmaceutically acceptable salts include the acid salts, for example, those of hydrochloric, citric, succinic, maleic, tartaric, acetic, and like acids. Of the acid salts, the acetate and hydrochloride are preferred. Such salts are pharmaceutically acceptable in the sense of having no substantially different activity or toxidity compared to the base.

The acetate and diacetate salts are especially preferred.

Solutions of the principal active ingredient as a free base or salt can be prepared in water or in water suitably mixed with, for example, surfactants. The preferred compound, where R is ethyl in the above formula, is slightly soluble in water. It can, for example, be converted to a partial acetate having a pH in aqueous solution of about 7.4 which, on analysis, shows about 1-1/2 acetic acid residues/anthraquinone nucleus. A diacetate may also be produced having a pH in aqueous solution of about 6.2. The diacetate is soluble in water to the extent of about 400 mg/millilitre of water. The base or various salts may be made more soluble by the addition of surfactants such as hydroxypropylcellulose to the composition. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorgansims.

The anthraquinones and their salts can be formulated in forms suitable for injectable use, which forms include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dis persion medium containing for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention ofthe action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example parabens, chlorobutanol, benzoalcohol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the principal active ingredient or ingredients in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freezedrying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The powders can also be sterilised by use of a gas, for example, ethylene oxide and subsequently incorporating, with the required additional ingredients and in the proper particle size, into the basic powder for later reconstitution with the desired suspending liquid which, of course, itself must be sterile.

It is especially advantageous to formulate the substances of this invention as pharmaceutical compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discreet units suited as unitary dosages for the animal and human subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.The specifications for the novel dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as disclosed in detail in this specification, these being features of the present invention.

The dosage of the principal active ingredient for the treatment of the indicated conditions depends upon age, weight and condition of the subject being treated; the particular condition and its severity; par ticularform of the active ingredient and the route of administration. A daily dose of from about 1 to about 100 mg/kg, given singly or in divided doses of up to 5 times a day embraces the effective range for the treatment of most conditions for which the compound is effective and substantially non-toxic. For a 75 kg subject, this translates into between about 75 and about 7500 mg/day. If the dosage is divided, for example, into 3 individual dosages, these will range from about 25 to about 2500 mg of the active ingredient. The preferred range is from about 2 to about 50 mg/kg of body weight/day with about 2 to about 30 mg/kg/day being more preferred.

The active ingredient of this invention is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically-acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active ingredient in amounts ranging from about 0.1 to about 400 mg, with from 1 to about 30 mg being preferred.

Expressed in proportions, the active ingredient is generally present in from about 0.1 to about 400 mg/ml of carrier.

In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Regression and palliation of cancers are attained, for example, using intraperitoneal administration. A single intravenous dosage or repeated daily dosages can be administered. Daily dosages up to about 5 to 10 days are often sufficient. It is also possible to dispense one daily dosage or one dose on alternate or less frequent days. As can be seen from the dosage regimens, the amount of principal active ingredient administered is a sufficient amount to aid regression and palliation of the leukaemia orthe like, in the absence of excessive deleterious side effects of a cytotoxic nature to the hosts harbouring the cancer.

Inthafollowing data using mice and the base of the above formula where R is ethyl, it can be seen that some toxicity exists at about 50 mg/kg and that increasing toxicity occurs from 50 to about 100 mg/kg. Nevertheless, daily dosages up to about 100 mg/kg are within the preferred range, especially when administered on different regimens and for various of the anthraquinone compounds and salts within the scope of the present invention.

As used herein, cancer is meant blood malignancies such as leukaemia, as well as other solid and non-solid malignancies such as the melancarcinomas, lung carcinomas and mammarytumours.

By regression and palliation is meant arresting or retarding the growth of the tumour or other manifestation of the disease compared to the course of the disease in the absence of treatment. In many of the following Examples, mice are used as standard test animals to show the effective and toxic levels of the novel compositions. From these data we infer that one can successfully treat humans with the sub stances of this invention to aid in the amelioration and regression of cancer diseases. From the test data with lower mammals it would be expected that the novel compositions are so effective below sub stantiallytoxic levels.

The following Examples set forth the manner and process of making and using the inventive composi tions. They also include test data showing the effec tiveness of the present compositions in treating leukaemia-like tumours in standard test animals.

Example 1-Preparation of Base 121 grams of para leuco quinizarine, or 2,3 dihydro - 1,4 - dihydroxy anthraquinone, were charged with 300 cc of ethyl alcohol solvent into a 1 litre flask. The mixture was agitated to form a uniform slurry, and to the slurry was charged 124 grams of N - aminoethylethanolamine. The resulting mixture was agitated at room temperature for one hour, heated to 75-80"C and held at that temperature for six hours. While the temperature was maintained at 75-80 C, the mixture was aerated until the leuco product was oxidised as indicated by the product being completely in solution. The solution was cooled to 10"C, filtered, washed with ethyl alcohol and dried under vacuum. The yield was 138 grams.

Other bases within the scope of this invention can be made with N - aminoethylethanolamine replaced by N - aminomethylmethanolamine, N - aminopropylpropanolamine or N - aminobutylbutanolamine.

Example 2 -- Preparation of Salts Any of the bases of Example 1 can be neutralised with an acid such as acetic or hydrochloric acid to form a salt. An excess of dilute aqueous acetic acid is added to the dried compound of Example 1 (derived from N - aminoethylethanolamine). The mixture is heated to about 50"C and held there for about 2 hours. An excess of chloroform is added and after phase separation, the organic phase is discarded.

The salt is then vacuum dried.

Example 3-Preparation oflnjectable Composition Ten grams of the acetate salt of Example 2 are dissolved in one litre of saline solution creating a solution with 10 mg/ml of active ingredient. A five ml dose of this composition thus provides 50 mg of active ingredient.

Solutions can be prepared of the various bases and salts of the invention with most standard pharmaceutically-acceptable solvents. Particularly with the free bases, a surfactant such as hydroxypropylcelluiose improves solubility.

Example 4 -- Preparation of Powder Composition Ten grams of the free base of Example 1, prepared using N - aminoethylethanolamine is dispersed in 190 grams of glycerine. An one gram dose of this powder thus provides 50 mg of active ingredient.

Powders may be prepared with most standard pharmaceutically-acceptable dispersion agents. The powder may then be combined with other agents as desired and encapsulated by conventional techniques.

Example 5 Six male mice of strain CDF1 were injected on day 0 intraperitoneally with 106 cells of L-1210 lymphoid leukemia. Beginning on day 1 and daily for a total of 9 days, each mouse was treated by an intraperitoneal injection of 250 mg/kg of body weight of the compound of Example 1 in a carrier of hydroxypropylcellulose sold as Kluccel. After five days two of the six have survived. The mean survival time was 6.5 days for the test animals, 9.9 days for the controls.

Examples 6 to 11 The test of Example 5 was repeated for groups of six mice and a group of controls at the doses indicated in Table 1. The means survival times are indicated in Table 1. The test was discontinued after 30 days.

Table 1 -LyphoidLeukemia (L-1210) Dose mglkg Survival Mean Body at 5 Survival Apparent Example Weight days Time Cures** 5 250 2/6 6.5 6 125 4/6 6.8 7 62.5 5/6 21.8 2 Control 0 9.9 95.0 6/6 7.3 62.5 6/6 11.3 42.0 6/6 11.3 11* 28.0 6/6 18.3 2 Control 0 8.4 * Mouse strain BDF,.

Mice surviving until end of test.

It appears from these tests that dosages of 62.5 mg/kg body weight and over begin to show some time toxicity to mice underthistest regime. Substantially improved survival times compared to the controls (innoculated only) is found in dosages from about 28 to about 62.5 mg/kg of body weight.

Example 12 Six male mice, strain CDF1, were innoculated with P388 lymphocytic leukemia applied intraperitoneally at 106 cells in ascitic fluid. Beginning the first day after innoculation and then daily for nine days, the compound of Example 1 was applied at a close level of 128 mg/kg of body weight in saline solution with "Tween-80". Four of six mice survived after five days and the median survival time for the six mice was 5.9 days.

Examples 13 to 29 Example 12 was repeated with groups of six mice at different dosage levels with the results shown in Table 2. This test was discontinued after 30 days.

Table 2 - Lymphocytic Leukemia (P388) Dose mglkg Survival Mean Body at 5 Survival Apparent Example weight days Time Cures 12 128 4/6 5.9 13 64 6/6 6.4 14 32 6/6 8.8 15 16.0 6/6 29.7 3 16 8.0 6/6 29.9 4 17 4.0 6/6 29.7 3 Control 0 10.8 18 128 6/6 6.4 19 64 4/6 6.3 20 32 6/6 7.8 21 16.0 6/6 10.1 22 8.0 6/6 21.0 23 4.0 6/6 29.7 3 Control 0 11.0 24 128 2/6 5.0 25 64 6/6 6.2 26 32 5/6 8.0 27 16.0 6/6 28.8 2 28 8.0 6/6 23.0 29 4.0 6/6 19.8 Control 0 10.8 NOTE: The "%" figure in this and subsequent Tables indicates that the only surviving animals were adjudged upon autopsy to be "no-takes" for the tumour.

These tests show substantial activity in the 4.0-16.0 mg/kg body weight range. At a dosage of 32.0 mg/kg and especially 64.0 mg/kg, the toxicity of the drug appears to overtake the beneficial effects.

Example 30 Ten more male mice, strain BDF1, were innoculated intraperitoneally with an unspecified level of the homogenate of tumour B-16 meloncarcinoma.

Beginning the first day after innoculation and every second day thereafter, the mice were treated with intrperitoneal injections of 128 mg/kg of body weight ofthe compound of Example 1 in saline solution with "Tween-80". Injections occurred intraperitoneally on survivors on days 3,5,7,9, 11, 13, 15 and 17.

The median survival time was 5.2 days compared to 21.3 days for the control. Evaluation ended on the 60th day.

Examples 31 to 47 Example 30 was repeated at dosage levels shown in Table 3 with the results indicated in Table 3.

Table 3 - Melancarcinoma (B- 16) Dose mglkg Survival Mean Body at 5 Survival Apparent Example Weight days Time Cures 30 128 3/10 5.2 31 64.0 8/10 6.1 32 32.0 10/10 10.8 33 16.0 10/10 59.8 8 34 8.0 10/10 59.8 6 35 4.0 10/10 59.8 6 Control 0 21.3 36 128 4/10 5.4 37 64.0 8/10 7.1 38 32.0 10/10 11.1 39 16.0 10/10 51.0 tumoured survivors only 40 8.0 10110 60.0 10 41 4.0 10/10 43.0 Control 0 25.3 42 128 10/10 9.3 43 64.0 8/10 16.8 44 32.0 10/10 37.0 45 16.0 10/10 34.0 46 8.0 10/10 36.0 47 4.0 10/10 32.0 Control 0 18.8 48** 128 7.0 49** 64.0 12.0 50** 32.0 31.0 51** 16.0 31.0 52** 8.0 26.3 53** 4.0 27.0 Control 0 30.3 * The serum of Examples 30 to 41 was diluted 10:1 before inocculation in Examples 42 to 53. Also the surfactant Tween-80 was omitted.

** In Examples 48 to 53, the injections were on days 1,3,5,7,9, 11, 13, 15 and 17.

Example 54 Six male mice, strain CDF1, were innoculated intraperitoneally with 105 cells of L-1 210 lymphoid leukemia. Beginning on the first day after innoculation and daily for a total of nine days, the mice were innoculated with intraperitoneal injections of 128 mg/kg of body weight of the compound of Example 1 in saline. Five of the six mice survived to the fifth day and the median survival time was 6.1 days. The test was terminated after 30 days.

Examples 55 to 57 Example 54 was repeated at the dosage levels indicated in Table 4. In examples 62 to 69 the injections were subcutaneous. In Examples 70 to 77 the compound was given Orally.

Table 4 - Lymphoid Leukemia (L- 1210) Dose mglkg Survival Mean Body at 5 Survival Apparent Example Weight days Time Cures 54lP 128 5/6 6.1 55 64.0 6/6 7.8 56 32.0 6/6 11.0 57 16.0 6/6 15.3 58 8.0 6/6 16.0 59 4.0 6/6 12.8 3 60 2.0 6/6 12.7 61 1.0 6/6 11.3 62 SC 128 6/6 10.3 3 63 64.0 6/6 11.8 64 32.0 6/6 11.8 65 16.0 6/6 10.4 66 8.0 6/6 8.4 67 4.0 6/6 8.3 3 68 2.0 6/6 8.4 69 1.0 6/6 8.2 70 Oral 512 6/6 8.1 71 256 6/6 9.0 72 128 6/6 8.8 73 64.0 6/6 8.8 74 32.0 6/6 8.4 75 16.0 6/6 8.4 76 8.0 6/6 9.2 77 4.0 6/6 9.2 Control 0 8.0 When given intraperitoneally, the compound showed substantial activity at dosages of 16.0 and 8.0 mg/kg of body weight.More limited activity was seen in this particular test series when the same or higher dosages were injected subcutaneously or were introduced by oral administration.

Example 78 Ten male mice, strain BDF1, were innoculated with Lewis lung carcinoma homogenate at unspecified dosage intravenously. Beginning on the first day after innoculation and for a total of nine days, the mice were treated with 32 mg of the compound of Example 1 per kg of body weight. After five days all 10 mice survived. The median survival time was 8.2 days.

Examples 79 to 89 Example 78 was repeated at the dosage levels shown in Table 5. In Examples 84 to 89, tumour fragments rather than tumour homogenate were used for innoculation. These tests were discontinued after 60 days.

Table 5-Lewis Lung Carcinoma Dose mglkg Survival Mean Body at 5 Survival Apparaent Example Weight days Time Cures 78 32.0 10/10 8.2 79 16.0 10/10 18.0 80 8.0 10/10 32.0 81 4.0 10/10 23.3 82 2.0 10/10 25.0 83 1.0 10/10 22.8 Control 0 22.7 84 32.0 10/10 8.4 85 16.0 10/10 11.0 86 8.0 10/10 28.0 87 4.0 10/10 27.0 88 2.0 10/10 39.0 89 1.0 10/10 50.3* Control 0 24.0 For this tumour, the effect was appreciably over the dosage range of 2.0-8.0 mg/kg although the results are not as dramatic as for othertumours. The high value in Example 89 is somewhat inconsistent with the toxicity pattern based on body weight change, which peaks at a dosage of 2.0 mg/kg.

Example 90 Six female mice, strain CDF1, were innoculated intraperitoneally with 106 cells of P388 lymphocrytic leukemia. On the first day after innoculation and on days 5 and 9 for a total of three days, the mice were treated with 512 mg/kg of body weight of the com pound of Example 1 in saline solution with "Tween-80" surfactant Tests were discontinued after 30 days. The median survival time was 2.1 days.

Examples 91 to 102 Example 90 was repeated at the dosage levels indicated in Table 6 with the results indicated.

Table 6-Female Mice Dose mglkg Survival Mean Body at 5 Survival Apparent Example Weight days Time Cures 90* 512 0/6 2.1 91* 256 0/6 2.7 92* 128 2/6 5.0 93* 64.0 5/6 7.8 94* 32.0 6/6 29.7 95* 16.0 6/6 26.0 Control 0 11.1 96** 64.0 4/6 6.3 97** 32.0 6/6 8.7 98** 16.0 6/6 34.9 4 99** 8.0 6/6 28.0 100** 4.0 6/6 22.3 101** 2.0 6/6 24.0 102** 1.0 5/6 17.9 Control 0 10.5 * Examples 90 to 95 involved injection on days 1, 5 and 9 only.

* * Examples 96 to 102 and their control were continued to the thirty-fifth day.

Injections were daily for nine days.

These tests show substantial activity, particularly at dosages of about 16.0 mg/kg.

Example 103 Ten male mice, strain BDF1, were innoculated with the same unspecified amount of B16 melanocar- cinoma tumour homogenate as in Examples 30 to 41, diluted 1:10 as in Examples 42 to 53, intraperitoneally. Beginning on the first day after innoculation, and every other day thereafter for a total of nine injections, the mice were injected with 125 mg/kg of body weight of the compound of Example 1 in saline solution with hydroxypropylcellulose, sold as Kluccel. All ten mice survived after 5 days. The median survival time was 8.4 days. The test was discontinued after sixty days.

Examples 104 to 107 Example 103 was repeated at the doses indicated in Table 7.

lable 7 Dose mglkg Survival Mean Body at5 Survival Apparent Example Weight days Time Cures 103 125 10/10 8.4 104 62.5 10/10 16.0 2 105 31.2 10/10 49.0 2 106 16.6 10/10 43.3 2 107 8.3 10/10 42.8 Control 0 18.7 In these tests, substantial results were shown over the dosage range of 8.3-31.2 mg/kg. These results further support the activity against melancarcinoma shown in Examples 30 to 53.

Example 108 Ten male mice, strain CDF1, were innoculated with a dilute homogenate of Colon 26 tumour (National Cancer Institute identification C6). They were then injected with 125 mg/kg of body weight of the compound of example 1 in hydroxypropylcellulose solution, sold as Kluccel, on days 1,5 and 9 after innoculation. This evaluation was continued for 70 days. All ten mice survived the fifth day and the median survival time was 27.0 days compared to 26.5 for the controls.

Examples 109 to 112 Example 108 was repeated at the dosages shown in Table 8.

Table 8 - Tumour C6 Dose mg/kg Survival Median Body at 5 Survival Apparent Example Weight days Time Cures 108 125 10/10 27.0 109 35.2 10/10 32.0 110 31.2 10/10 29.0 111 16.6 10/10 29.0 112 8.3 10/10 33.8 Control 0 26.5 These tests show some effectiveness against this particulartumour. A lesser degree of toxicity was found at the 125 mg/kg level compared to some of the other Examples.

Example 113 Ten male mice, strain CD8F1, were innoculated with CD8F1 mammary tumour homogenate between about 500 to 1000 mg in size, subcutaneously. On days 1,8, 15 and 22 after innoculation, the mice were treated with an injection of 125 mg/kg of body weight of the compound of Example 1 in hydroxypropylcellulose (Kluccel) intraperitoneally. Eight of ten mice survived the fifth day. After 36 days, the mice were killed and the mean tumour weight was determined to be 630 mg.

Examples 114 to 117 Example 113 was repeated at the dosages shown in Table 8.

Table 8 - Mammary Tumour CD8F1 - Tumour Weight Dose Mean mglkg Survival Tumour Body at5 Weight Example Weight days (mug) 113 125 8/10 630 114 62.5 9/10 989 115 31.2 10/10 423 116 16.6 10/10 1354 117 8.3 35/35 37 Control 0 1665 These results indicate substantial inhibition of tumour growth at many levels and apparent decreases in tumour sizes in some instances.

Claims (6)

1. For use in the treatment of the human body for therapy of cancer, an anthraquinone of formula:

wherein R is an alkylene group having 1 to 4 carbon atoms.

2. The anthraquinone of formula I, for anti-cancer use, wherein R is ethylene.

3. Pharmaceutically acceptable salts of the anthraquinones defined in claim 1 or 2.

4. A salt according to claim 3, which is an acetate salt.

5. A salt according to claim 3, which is diacetate salt.

6. An anthraquinone or salt thereof according to any preceding claim, for use in the treatment of the human body for therapy of leukaemia.