NO155088B - PROCEDURE FOR AA IMPROVE STORAGE STABILITY OF AN N4 ACYLCYTOSINARABINOSIDE. - Google Patents

Description

The present invention relates to a method for improving the storage stability of an N 4-acylcytosine arabinoside, which is useful in the treatment of cancer.

As shown in US Patent No. 3,991,045, an N 4 -acylcytosine arabinoside is a useful agent in the treatment of cancer, is particularly effective against leukemia in mice, and exhibits resistance to deaminase inactivation.

However, when an N 4 -acylcytosine arabinoside is stored as is, at room temperature or more stringent conditions, it breaks down to form its deacylated product and loses its activity. Therefore, when preparing orally ingestible medicines containing an N 4 -acylcytosine arabinoside, it is necessary to prevent the cleavage reaction.

Antioxidants, pH-regulating agents, excipients etc. such as. usually used to improve the chemical stability is not able to prevent cleavage of N 4-acylcytosine-arabinoside.

It is therefore desirable to develop a drug containing an N 4 -acylcytosine arabinoside with excellent storage stability.

It has been found that it is possible to stabilize an N 4-acylcytosine arabinoside, so that this can be utilized in practice, by adding to it at least one monoglyceride of an aliphatic acid with a hydrophobic long-chain aliphatic acid group and a nonionic surfactant with a hydrophilic polyoxyethylene group as a side chain whereby an active mixture is obtained in which the N 4-acylcytosine-arabinoside is stabilized to a significant extent.

100 parts by weight of N<4->acylcytosine-arabinoside added with 10 - 100 parts by weight of a monoglyceride of an aliphatic acid and/-

or 5-500 parts by weight of a non-ionic surfactant with a polyoxyethyl group as side chain, can improve the stability of the method according to the invention. By mixing the nonionic surfactant with a

hydrophilic polyoxyethylene group as a side chain, an N 4-acylcytosine-arabinoside will be markedly stabilized, and even after 30 days from the beginning of a storage experiment at 50°C, almost no cleavage of the N 4-acylcytosine-arabinoside could be detected.

According to the invention, the non-ionic surfactant is used in an amount of 5 - 500 parts by weight, preferably 10 - 200 parts by weight per 100 parts by weight of the N 4 -acylcytosine arabinoside. In amounts of less than 5 parts by weight, the N 4 -acylcytosine arabinoside will not be stabilized. On the other hand, amounts greater than 500 parts by weight will cause the desired powder properties for the production of an orally ingestible medicine to be lost.

When a monoglyceride of an aliphatic acid with a hydrophobic long-chain aliphatic acid group is added, an N 4-acylcytosine-arabinoside is markedly stabilized, and even after 90 days after the initial storage experiment at 50°C, almost no cleavage of the N 4-acylcytosine-arabinoside can be detected . By adding smaller amounts of the aliphatic acid monoglyceride, no stabilizing effect is obtained. On the other hand, the addition of larger amounts of the monoglyceride will not give any increase in the stabilizing effect and is also not preferred from an economic point of view. The aliphatic acid monoglyceride is therefore added in an amount of 10 - 100 parts by weight per 100 parts by weight of N 4-acylcytosine-arabinoside.

The monoglyceride of this aliphatic acid is very effective in stabilizing the N 4 -acylcytosine-arabinoside, but lowers its pharmaceutical effect somewhat. It has therefore been found that this disadvantage can be removed by both adding the non-ionic surfactant and the monoglyceride of the aliphatic acid. It is thus particularly preferred to mix both the non-ionic surfactant and the monoglyceride of the aliphatic acid, whereby excellent stability is obtained without reducing the pharmaceutical effect and the amounts of the non-ionic surfactant and the monoglyceride can be reduced. When such a mixture is kept at 50 C for 90 days, almost no cleavage of the N 4-acylcytosine-arabinoside can be detected and no reduction in the pharmaceutical effect can be observed. In this case, it is preferred to add 5-50 parts by weight of the monoglyceride of the aliphatic acid and 5-100 parts by weight of the non-ionic surfactant per 100 parts by weight of the N 4-acylcytosine arabinoside.

The monoglyceride of the aliphatic acid used in the present invention has generally previously been used as an emulsifier for foodstuffs or cream base for cosmetics or medicines. The monoglyceride of the aliphatic acid has therefore previously been used for stabilizing an emulsion but has not previously been used for chemical stabilization of a pharmaceutical active ingredient.

The nonionic surfactant with a polyoxyethylene group as a side chain has wide application as a solubilizing agent, emulsifying agent, suspending agent and the like. However, it is not previously known that the nonionic surfactant effects a stabilization of an N 4 -acylcytosine arabinoside, i.e. that it has the effect of inhibiting the release and transfer of the acyl group in the N 4-acylcytosine-arabinoside.

Although anionic surfactants such as sodium dodecyl sulfate exhibit a stabilizing effect, it is insufficient in practice. Furthermore, as it is generally believed that water participates in the release and transfer reaction for the acyl group, carriers with a low water content and additives with hygroscopic and moisture-retaining properties such as mannitol, fine crystalline cellulose, anhydrous silicon dioxide, synthetic aluminum silicate etc. have been investigated. However, they have been found to have no positive stabilizing effect for N 4 -acylcytosine arabinoside. N 4-acylcytosine arabinosides which can be used according to the invention as the active ingredient include those which have. an aliphatic acyl group with 3-24 carbon atoms in the N 4 position, such as N 4 -propionylcytosine arabinoside, N 4 -butyrylcytosine arabinoside, N 4 -valerylcytosine arabinoside, N 4 -caproylcytosine arabinoside, N 4 -heptanoylcytosine arabinoside, N 4 -caprylylcytosine arabinoside, - N 4 -caprylcytosine arabinoside, N 4 -lauroylcytosine arabinoside, N 4 -myristoylcytosine arabinoside, N 4 -pentadecanoylcytosine arabinoside, N 4 -palmitoylcytosine arabinoside arabinoside, N 4 -margaroylcytosine arabinoside, N 4 -stearoylcytosine arabinoside', N 4 -nonadecanoylcytosine arabinoside, N 4 -arachidoyl cytosine arabinoside, N 4 -heneicosanoylcytosine arabinoside, N 4 -behenoylcytosine arabinoside., N 4 -tricosanoylcytosine arabinoside, N 4 -lignoceroylcytosine arabinoside, N 4 -palmitoleylcytosine arabinoside, N 4 -oleoylcytosine arabinoside, N 4 -elaidoylcytosine arabinoside, N 4 -vaccenoylcytosine arabinoside, N 4 -linoleylcytosine arabinoside, N 4 4-linoleylcytosine arabinoside, N 4-rachidonoylcytosine arabinoside, etc.

Monoglycerides of aliphatic acids that can be used according to the invention include those derived from a higher aliphatic acid with 12 - 18 carbon atoms and glycerol, such as stearic acid monoglyceride, palmitic acid monoglyceride, myristic acid monoglyceride, lauric acid monoglyceride, etc. These monoglycerides can be used alone or in combination with each other.

Examples of non-ionic surfactants with a polyoxyethylene group as side chain or chains that can be used according to the invention include polyoxyethylene aliphatic acid esters (preferably those to which an average of 20 - 100 mol of oxyethylene units are added and more preferably stearates to which an average of 20 - 55 mol oxyethylene units), polyoxyethylene castor oil or hydrogenated castor oil (in both cases they are to which 20 - 100 mol oxyethylene units are added on average), polyoxyethylene glycerol esters of aliphatic acids or polyoxyethylene propylene glycol esters of aliphatic acids (especially preferred are monostearates or oleates to which are added on average 10 - 60 mol oxyethylene units), polyoxyethylene lanolin or beeswax derivatives (especially preferred are those to which an average of 30 - 100 mol oxyethylene units have been added), polyoxyethylene sorbitan esters of aliphatic acids and polyoxyethylene sorbitol esters of aliphatic e. acids, polyoxyethylene alkyl ethers (especially preferred are those to which an average of 20 - 60 mol oxyethylene units have been added), polyoxyethylene alkylphenyl ethers (especially preferred are those to which an average of 10 - 60 mol oxyethylene units have been added), polyoxyethylene-polyoxypropylene alkyl ethers (especially preferred are those to to which are added an average of 20 - 60 mol oxyethylene units plus oxypropylene units), polyoxyethylene-polyoxypropylene condensates (especially preferred are those with an average molecular weight of 2,000 - 12,000 and which contain 30 - 90% by weight of oxyethylene units), etc. These non-ionic surfactants can be used alone or in combination with each other.

Of these compounds, polyoxyethylene aliphatic acid esters, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene glycerol esters of aliphatic acids, polyoxyethylene propylene glycol esters of aliphatic acids, polyoxyethylene lanolin, polyoxyethylene beeswax, polyoxyethylene sorbitan esters of aliphatic acids and polyoxyethylene sorbitol esters of aliphatic acids are particularly preferred as they give a very great stabilization effect in relatively small quantities.

The most preferred compounds are polyoxyethylene aliphatic acid esters, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene glycerol esters of aliphatic acids and polyoxyethylene propylene glycol esters of aliphatic acids. When these are used in combination with aliphatic acid monoglycerides, they provide good results.

When the nonionic surfactants are added alone to an N 4 -acylcytosine arabinoside, the acceptable maximum amount of the surfactant is usually 50 parts by weight

per 100 parts by weight of N 4-acylcytosine-arabinoside so that the mix-

one must be present as a powder. When the surfactant is used in larger amounts than 50 parts by weight, it is necessary to add excipients with a low water content, for example mannitol and additives that have previously been dehydrated

hardened and dried, for example fine crystalline cellulose, anhydrous silicon oxide, metasilicic acid-magnesium aluminate, synthetic aluminum silicate, starch, hydroxypropyl starch etc. alone or in combination to the obtained oily or waxy mixture of N 4-acylcytosine-arabinoside and the surfactant, or that these additives are added beforehand to the N 4-acylcytosine-arabinoside, after which the surfactant is mixed with the resulting mixture.

When an improved wetting and additional stability is necessary for the mixture prepared according to the invention,

common anionic surfactants can be added. However, as these anionic surfactants cause side effects, for example irritation of the digestive tract, it is desirable that the added amount be kept below 50 parts by weight per 100 parts by weight N 4-acylcytosine-arabinoside.

Suitable examples of such anionic surfactants include sodium lauryl sulfate, sodium deoxycholate, sodium dioctyl sulfosuccinate, and the corresponding potassium salts thereof.

The mixing method for the ingredients can be achieved according to

known technique, for example a wet method in which the non-ionic surfactant and/or the monoglyceride of the aliphatic acid is dissolved in a low-boiling solvent, for example an alcohol, ester, ketone, ether or the like,

and if necessary during heating, after which the obtained solution is added to the N 4-acylcytosine-arabinoside or a mixture thereof with an excipient and then stirred and kneaded or dissolved, after which the solvent is removed and the thus obtained solid is pulverized and sieved. The components can be brought together using a dry method whereby the nonionic surfactant and/or the aliphatic

acid monoglyceride is pre-pulverized and the N 4-acylcytosine-arabinocide or a mixture thereof with an excipient bg is added, if necessary, a lubricant is added after which they are mixed and pulverized using a measuring device such as a ball mill, a vibrating ball mill, stick mill, atomizer, jet mixer, mortar , etc. Furthermore, these methods can also be used in combination.

The following examples illustrate the invention.

The storage stability of mixtures prepared according to the invention was measured in the following way: the mixture was allowed to stand at 50°C for a predetermined period of time and the remaining N 4 -acylcytosine arabinoside was determined by means of liquid chromatography and the storage stability is indicated as the remaining part (% by weight) of the active ingredient (N 4-acylcytosine-arabinoside).

EXAMPLE 1

3 g of polyoxyethylene (40) monostearate [the number in parentheses indicates the average number of moles of oxyethylene units added, hereinafter the number will have the same meaning] ("MYS-40" manufactured by Nikko Chemicals Co., Ltd.) was dissolved in 20 ml of ethanol under heating. The obtained solution was added to 10 g of N 4 -stearoylcytosine-arabinoside and the mixture was carefully stirred and kneaded in a mortar and then vacuum dried. The solid thus obtained was pressed through a 60 mesh sieve.

EXAMPLE 2

A mixture of 1 g of polyoxyethylene (60) hydrogenated castor oil ("HCO-60" manufactured by Nikko Chemicals Co., Ltd.) and 1 g of polyoxyethylene (40) monostearate ("MYS-40") was dissolved in 20 ml of ethanol under heating. The obtained solution was added to 10 g of N 4 -palmitoylcytosine-arabinoside and then stirred and pounded in a mortar and then vacuum dried. The resulting solid was pressed through a 30 mesh sieve. A mixture of 5 g mannitol and 3 g dry silica was added and the mixture pulverized in a ball mill for 1 h.

EXAMPLE 3

2 g of polyoxyethylene (40) monostearate ("MYS-40") which was coarsely pulverized at low temperature and passed through a 60 mesh sieve was added to 10 g of N 4 -behenoylcytosine arabinoside and mixed and pulverized in a ball mill for 30 min .

EXAMPLE 4

10 g of polyoxyethylene (60) hydrogenated castor oil ("HCO-60") was dissolved in 100 ml of ethanol under heating. The obtained solution was added to a mixture of 10 g of N 4 -myrisoyl-cytosine-arabinoside and 40 g of mannitol and then stirred and pounded in a mortar and then vacuum dried. The solids obtained were pressed through a 30 mesh sieve and pulverized in a ball mill for 1 h.

EXAMPLE 5

5 g of polyoxyethylene (40) monostearate ("MYS-40") was dissolved in 50 ml of ethanol under heating. The resulting solution was added to 10 g of N 4 -pentadecanoylcytosine-arabinoside and then stirred and pounded in a mortar and then allowed to stand overnight at room temperature. To this sticky mixture in which a small amount of the solvent was present, 10 g of dry silica was added and then mixed and pulverized until a powdery mixture was obtained which was vacuum dried. The solid thus obtained was pressed through a 60 mesh sieve.

The powders obtained according to examples 1-5 were subjected to a storage test at a temperature of 50°C for 30 days and the remaining part of the active ingredient was determined. For comparison, the active ingredients used in examples 1-5 alone were examined in the same way. The results are shown in table 1.

EXAMPLES 6-22

The procedure according to examples 2, 3, 4 or 5 was repeated but where the N 4 -acylcytosine arabinoside and the additives were changed. The solids thus obtained were subjected to storage tests at 50°C for 30 days and the remaining part of the active ingredient was determined. The results are shown in table 2. All conditions are per weight.

EXAMPLE 23

Polyoxyethylene (40) monostearate ("MYS-40"), manufactured by Nikko Chemical Co., Ltd) was added to 100 ml of ethanol in amounts shown in Table 3 and dissolved by heating.

The solutions thus obtained were added to a mixture of 10 g of N 4 -stearoylcytosine-arabinoside and 40 g of mannitol and then stirred and pounded in a mortar and vacuum dried. The solids obtained were pressed through a 30 mesh sieve and then pulverized in a ball mill for 1 h.

EXAMPLE 24

A mixture of 3 g of N 4 -stearoylcytosine arabinoside, 1.5 g of stearic acid monoglyceride and 7.5 g of mannitol (excipient) was milled in a vibrating ball mill for 3 h to give a dry powder.

The remaining part of the active ingredient after storage at 50°C for 90 days, determined as the percentage of the original amount present, was 99.5%.

On the other hand, when N 4-steorylcytosine-arabinoside as such was stored under the same conditions, the remaining proportion of the active ingredient was 89.0%.

EXAMPLE 2 5

A mixture of 3.0 g of N 4 -stearoylcytosine arabinoside and 1.5 g of stearic acid monoglyceride was dissolved in 300 ml of ethanol under heating. The ethanol was then removed under reduced pressure. To the powder obtained, 7.50 g of mannitol was added and the mixture was ground in a vibrating ball mill for 3 hours to give a powder which, after storage at 50°C for 90 days, contained 99.6% of the active ingredient.

EXAMPLE 26

A mixture of 3.0 g of N 4 -palmitoylcytosine arabinoside, 1.5 g of stearic acid monoglyceride and 7.5 g of mannitol was milled in a vibrating ball mill for 3 h to give a powdery mixture.

The remaining proportion of the active ingredient after storage at 50 o C for 90 days was 99.4%, while for N 4-palmitoylcytosine-arabinoside as such the remaining proportion was 88.0%.

EXAMPLE 27

A mixture of 10 g of N 4 -stearoylcytosine arabinoside and stearic acid monoglyceride in the amounts shown in Table 4 was milled in a vibrating ball mill for 3 h to give a powdery mixture whose remaining proportion of the active ingredient after storage at 50 °C for 90 days is shown in table 4.

EXAMPLE 28

A mixture of 5 g of N 4 -stearoylcytosine arabinoside and 2.5 g of each of the aliphatic acid monoglycerides shown in Table 5 or 2.5 g of polyoxyethylene (40) monostearate ("MYS-40" manufactured by Nikko Chemicals Co., Ltd.) and 12.5 g of mannitol were ground in a vibrating ball mill for 3 h to give a powdery product. The remaining proportion of the active ingredient when stored at 50°C for 90 days is shown in table 5.

EXAMPLE 2 9

A mixture of 2.5 g of stearic acid monoglyceride and 5 g of each of the N 4 -acylcytosine arabinosides shown in Table 6 and 12.5 g of mannitol was milled in a vibrating ball mill for 3 h to give a powdery product. The remaining component of the active component after storage at 50°C for 90 days is shown in table 6.

EXAMPLE 3 0

A mixture of 10.0 g of N 4 -stearoylcytosine arabinoside and stearic acid monoglyceride and polyoxyethylene stearate ("MYS-40", manufactured by Nikko Chemicals Co., Ltd.) in the amounts shown in Table 7 was dissolved in 1500 ml of ethanol while heating to 50°C. The ethanol was removed by evaporation under reduced pressure in a rotary evaporator. The solid obtained was pulverized in a mortar and in each case a fine powder with excellent flowability was obtained. A portion of the fine powder was allowed to stand at 50°C for 3 months and stability with time determined. To the remaining portion was added an appropriate amount of water and pounded in a mortar to give a dispersion with a concentration of N 4 -stearoylcytosine arabinoside of 30 mg/ml. This dispersion was investigated with regard to anticancer activity using mice as experimental animals.

To investigate the stability with time, the content of the remaining N 4 -stearoylcytosine-arabinoside after storage at 50°C for 3 months was determined and the stability was indicated as the remaining part of the active ingredient.

Anti-cancer activity tests were carried out in the following manner: Each mixture was administered directly into the stomach of a group of mice of 10 mice of the CDF line where each mouse was intra-peritoneally injected with 10 L-1210 mouse leukemia cells. N 4-steorylcytosine-arabinose in doses of 200 mg/kg was introduced once per day for 5 days using a catheter.

The average survival days (T) of mice in the experimental groups and the average survival days (C) of control mice that had not been given the active ingredient were determined and the anticancer activity of the mixture was expressed as a percentage of T bases on C (T/C%).

As it was not possible to prepare an aqueous dispersion of N 4-stearoylcytosine-arabinoside as such, it was dispersed

in a 0.5% "Tween 80" (polyoxyethylene sorbitan monooleate) aqueous solution and administered in this form (experiment no. 1 only).

The results are shown in table 7.

EXAMPLE 31

— 4

A mixture of 10.0 g of N -stearoylcytosine arabinoside, 2.5 g of each of the aliphatic acid monoglycerides shown in Table 8 and 1.0 g of polyoxyethylene stearate ("MYS-40") was dissolved in 1500 ml of ethanol while heating at 50°C. The ethanol was then removed by evaporation under reduced pressure in a rotary evaporator. The resulting solid was pulverized in a mortar and a fine powder with excellent flow properties was obtained.

The thus obtained fine powder was subjected to the same test as in example 30 and the results are shown in table 8.

EXAMPLE 32

10.0 g of N 4 -stearoylcytosine arabinoside, 2.5 g of stearic acid monoglyceride, 2.5 g of each of the nonionic surfactants containing the number of oxyethylene units shown in Table 9, as well as 26.5 g of mannitol (excipient) was mixed and pulverized in a vibrating ball mill for 1 h. The powder thus obtained was subjected to the same tests as stated in example 30 and the results are shown in table 9.

EXAMPLE 3 3

A mixture of 10.0 g of each of the N 4 -acylcytosine arabinosides shown in Table 10, 2.5 g of stearic acid monoglyceride and 1.0 g of polyoxyethylene stearate ("MYS-40") was dissolved in 1500 ml of ethanol by heating to 50°C. The ethanol was removed by evaporation under reduced pressure in a rotary evaporator. The solid thus obtained was pulverized in a mortar to give a fine powder. The thus obtained fine powder was subjected to the same stability test as in example 30 and the results are shown in table 10 together with the relevant N 4 -acylcytosine arabinosides as such.

EXAMPLE 3 4

10.0 g of N 4 -palmitoylcytosine arabinoside, 1.5 g of palmitic acid monoglyceride, 1.0 g of polyoxyethylene stearate ("MYS-4 0"), 0.5 g of each of the anionic surfactants shown in Table 11 and 27.0 g of mannitol (excipient) was mixed and pulverized in a vibrating ball mill for 1 h.

The powder thus obtained was subjected to the same tests as stated in example 30 and the results are shown in table 11.

EXAMPLE 3 5

10.0 g N 4-stearoylcytosine-arabinoside, 1.5 g stearic acid mono-

glyceride, 1.0 g of polyoxyethylene stearate ("MYS-40"), 0.5 g of each of the anionic surfactants shown in Table 12 and 27.0 g of mannitol were mixed and pulverized in a vibrating ball mill for 1 h.

The powder thus obtained was subjected to the same test as stated in example 30 and the results are shown in table 12.

Claims (1)

1. Method for improving the chemical stability and storage stability of an N 4-acylcytosine-arabinoside where the acyl group contains 3-24 carbon atoms, characterized in that 100 parts by weight of the N 4-acylcytosine-arabinoside are mixed with 10-100 parts by weight of a monoglyceride of an aliphatic acid and/or 5-500 parts by weight of a non-ionic surfactant containing a polyoxyethylene group as a side chain.