Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BACKGROUND OF l`HE INVENTION
1 Field of the Invention
This invention relates to water-soluble compositions
comprising N4-acylcytosine arabinosides which are useful as
medicines, and to a process for preparation thereof.
2. Description of the Prior Art
Cytosine arabinoside has been sold as a medicine, but its
pharmaceutical effect does not last long. Attempts have,
therefore, been made to acylate its N4-position. Tests on mice
intraperitoneally injected with N4-acylcytosine arabinosides
show that the duration of the pharmacological activity of
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N4-acylcytosine arabinosides against intraperitoneal diseases
is improved and the acylcytosine arabinosides retain resistance
to the enzymes which deactivate the cytosine arabinoside.
This effect of improvement differs depending upon the number of
carbon atoms in the acyl group of the N4-acylcytosine arabinosides.
It is known that the improving effect is generally higher when
the number of carbon atoms in the acyl group becomes 5 or more,
and is especially ou~standing when the acyl group contains 14
to 24 carbon atoms. On the other hand, however, when N4-
acylcytosine arabinosides having 5 or more carbon atoms in the
acyl group are used as orally administrable agents, vascular
injections such as intravenous injections, and external agents
such as suppositories, their effects are inferior to the effect
observed in intraperitoneal injection probably because the
efficiency of utilization by the living body is poor.
SUMMARY OF THE INVENTION
This invention provides a water-soluble composition
comprising (1) an N4-acylcytosine arabinoside and (2) at least
one additive selected from the group consisting of (a) polyoxyethylene-
bonded hydroxy fatty acid glycerin esters, (b) polyoxyethylene fatty
acid esters, (c) polyoxyethylene-bonded lanolin and (d) bile
acids, and a process for preparation thereof. Especially preferably,
the invention provides a composition of the above ingredients,
which further comprises (3) at least one auxiliary additive
selected from the group consisting of saccharides, aliphatic
polyols, inorganic chlorides, inorganic bromides, inorganic
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sulfates an~ aromatic carboxylic acids or salts thereof, and
a process for preparation thereof.
DETAILED DESCRIPTION OF T~IE INVFNTION
N4-Acylcytosine arabinosides become more difficult to
dissolve in water as ~he number of carbon atoms in the acyl group
increases. Those containing 5 or more carbon atoms in the acyl
group are very sparingly soluble in water, and this property
becomes especially outstanding with compounds having 14 or more
carbon atoms.
It is one of the objects of this invention to solubilize
N4-acylcytosine arabinosides by adding specified additives.
Another object of this invention is to increase the rate
of biological utilization of N4-acylcytosine arabinosides.
Generally, when medicines not appreciably soluble under
standard conditions are used together with surface active agents,
the rate of their dissolving, their dispersibility in the blood
and the angle of their contact with the skin or mucous membrane
OT the wall of the alimentary canal increase, but the rate of
their absorption from the skin, mucous membrane or the wall of
the alimentary canal decreases, and their concentration in the
blood is reduced by the metabolism or excretion through the
kidneys, etc. It has been believed, therefore, that their
effect against the peritoneal lesions by oral administration
or intravenous injection is inferior to intraperitoneal
injection that directly acts on the lesions.
The present inventor has now found that compositions
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of N4-acylcytosinc arabinosidcs containing specified additives
become water-soluble, and their rate of biological utilization
increases even when they are used as peroral agents, vascular
injections such as intravenous injections and external agents,
The present invention is directed to a ~later-soluble composi-
tion at least comprising (1) an N4-acylcytosine arabinoside and
(2) at least one additive selected from the group consisting
of (a) polyoxyethylene-bonded hydroxy fatty acid glycerin esters,
(b) polyoxyethylene fatty acid esters, (c) polyoxyethylene-bonded
lanoline and (d) bile acids, and a process for preparation
thereof. Preferably, the invention provides a water-soluble
composition comprising (1) an N4-acylcytosyne arabinoside,
(2) at least one additive selected from the group consisting
of (a) polyoxyethylene-bonded hydroxy fatty acid glycerin
esters, ~b) polyoxyethylene fatty acid esters, (c) polyoxyethylene-
bonded lanolin and ~d) bile acids, and (3) at least one auxiliary
additive selected from the group consisting of saccharides,
aliphatic polyols, inorganic chlorides, inorganic bromides,
inorganic sulfates and aromatic carboxylic acids or their salts,
and a process for preparation thereof.
The composition comprising an N4-acylcytosine arabinoside
and the above-mentioned additive is sold as a solid, and can be
used after adding an aqueous solution of an auxiliary additive
prior to use. The composition which further contains the
auxiliary additive in addition to the above-described additive
can be formulated preferably into a solid. The solid can very
simply form a stable liquid by merely mixing it with water prior
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to use. Such compositions achieve the objccts and advantages
of the present invention
The composition containing the N4-acylcytosine arabinoside
and the additive must be heated to 60 - 100C when it is dissolved
in an aqueous solution of an auxiliary additive prior to use.
However, the composition which contains the auxiliary additive
in advance readily dissolves in water at room temperature, and
is more preferred.
The additive used in this invention is at least one
compound selected from the group consisting of (a) polyoxyethylene-
bonded hydroxy fatty acid glycerin esters, (b) polyoxyethylene
fatty acid esters,(c) polyoxyethylene-bonded lanolin and (d)
bile acids. When the additive contains a polyoxyethylene group
(in the case of (a), (b) and (c)), it is preferred that 10 to
200 moles, preferably 20 to 100 moles, on an average, of
oxyethylene be polymerized at the polyoxyethylene portion.
When the additive contains a fatty acid residue (in the case
of (b)) or a hydroxy fatty acid residue (in the case of (a)),
fatty acid residues or hydroxy fatty acid residues containing
10 to 20 carbon atoms (e.g., capric acid, hydroxycapric acid,
stearic acid, hydroxystearic acid, eicosanoic acid, hydroxy-
eicosanoic acid, etc.) are preferred.
Of the additives used in the present invention, bile
acids (d) and glycerin esters of hydroxy fatty acids containing
10 to 20 carbon atoms (e.g., hydroxycapric acid, hydroxylauric
acid, hydroxymyristic acid, hydroxypalmitic acid, hydroxymargaric
acid, hydroxystearic acid, hydroxyeicosanoic acid, ricinoleic
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acid, etc.~ to which polyoxycthylene having an avcrage degrec
of polymerization of 10 to 200 moles, prcferably 40 to 100
moles bonds, are especifically preferred. Of the latter,
hydrogenated castor oil having added thereto polyoxyethylene
having an average degree of polymerization of 40 to 100 moles
and a glycerin ester of hydroxystearic acid having bonded thereto
polyoxyethylene having an average degree of polymerization of
40 to 100 moles are more preferred.
The bile acids denote bile acids and their salts, and
include, for example, such bile acids as deoxycholic acid,
dehydrocholic acid, cholic acid, lithocholic acid, chenode-
oxycholic acid, lagodeoxycholic acid, hyocholic acid and
phocaecholic acid, and salts of these bile acids. Of these,
the deoxycholic acid and dehydrocholic acid, and salts of these
are especially preferred. As the salts of bile acids, alkali
metal salts and ammonium salts are preferred. The sodium salts,
potassium salts, ammonium salts, trimethylammonium salts and
procaine salts are more preferred. The sodium salts are especially
superior. Especially preferred bile acids are sodium deoxycholate
and sodium dehydrocholate.
Accordingly, preferred additives are sodium deoxycholate,
sodium dehydrocholate, hydrogenated castor oil having bonded
thereto oxyethylene with an average degree of polymerization of
40 to 100 moles, and a glycerin ester of hydroxy stearic acid
having bonded thereto oxyethylene having an average degree of
polymerization of 40 to 100 moles. The hydrogenated castor oil
having bonded thereto the oxyethylene is most preferred.
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The xuxiliary ad~itive is at least one compound selerted
from sacchari~lcs, aliphatic polyols, inorganic chlorides, irlorgallic
bromidcs, inorganic sulfates and aromat~ic carboxylic acids or their
salts.
Preferred saccharides are monosaccharides such as ribose,
arabinose, glucose, fructose, sorbitol, mannitol, xylose, and
galactose, disaccharides such as lactose and saccharose and
in oral administration only, polysaccharides such as cellulose.
Glucose is most preferred.
Examples of the aliphatic polyols are glycerol, propylene
glycol and polyethylene glycol. The glycerol and propylene
glycol are especially preferred.
Preferred inorganic chlorides or bromides are chlorides
of alkali metals or alkaline earth metals, and bromides of alkali
metals or alkaline earth metals. Especially preferred chlorides
are NaCl, CaC12 and MgC12, and next comes KCl. On the other hand,
MgSO4 is especially preferred as the inorganic sulfate.
The aromatic carboxylic acids are, for example, ben~oic
acid and salicyclic acid, and are preferably used as alkali metal
salts, alkaline earth metal salts and ammonium salts. The sodium
salts are especially preferred, and sodium salicylate is the best
among them.
The amount of the additive or the auxiliary additive is
preferably 0.1 to 20 parts by weight per part by weight of the
N4-acylcytosine arabinoside. If the amount is less than 0.1
part by weight, the effect desired is not obtained. On the
other hand, amounts exceeding 20 parts by weight do not produce
any special result, and are not economical because the concent-
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ration of the N4-acylcytosine arabinoside decreases.
The co~position containing the N4-acylcytosine arabinoside
and the additive can be prepared by uniformly mixing these
ingredients. In order to mix them uniformly, it is the general
practice tG dissolve them in a solvent, which solvent is then
evaporated off from the solution. This results in the formation
of a composition consisting of the N4-acylcytosine arabinoside
and the additive. The composition consisting of the N4-acylcytosine
arabinoside and the additive is sold as a solid, and can be
used after addition of an aqueous solution of an auxiliary
additive prior to use.
The composition consisting of the N4-acylcytosine
arabinoside, the additive and the auxiliary additive can be
prepared by uniformly mixing these three ingredients.
Preferably, it is prepared by adding an aqueous solution of
the auxiliary additive to a composition consisting of the N4-
acylcytosine arabinoside and the additive. When water is
evaporated after forming the solution, the composition becomes
solid and is easy to transport. Lyophilization, however, is
especially preferred to render this composition solid.
Such a solid composition very simply becomes a stable liquid
by merely mixing it with water prior to use, and can achieve
the objects and advantages of the present invention.
In other words, in order to solubilize the N4-acylcytosine
arabinoside so as to meet the object of this invention, it is
only sufficient to mix the N4-acylcytosine arabinoside and the
additive, preferably further adding the auxiliary additive.
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For uniform mixing, it is the gencral practice to dissolve
the N4-acylcytosine arabinoside and the additive in a solvent,
followed by evaporating the solvent from the solution.
Preferably the auxiliary additive and water, or an aqueous
solution of the auxiliary additive, is added to the composition
consisting of the N4-acylcytosine arabinoside and the additive,
and the mixture is heated to form a solution, followed, if desired,
by evaporating the water from the solution.
In dissolving the N4-acylcytosine arabinoside and the
additive, the amount of the solvent used is not particularly
critical, but generally, ranges from 10 to 1,000 parts by weight
per part by weight of the N4-acylcytosine arabinoside.
The amount of water used together with the auxiliary additive
is neither restricted. Generally, the amount is preferably
S0 to 1,000 parts by weight per part by weight of the N4-
acylcytosine arabinoside.
In uniformly mixing the N4-acylcytosine arabinoside and
the additive and preferably further with the auxiliary additive,
the pressure, temperature and time conditions shown below can
be used. The N4-acylcytosine arabinoside and additive are
dissolved in a solvent for these materials at a pressure of
1 to 2 atms. and at a temperature of -20C to the boiling point
of the solvent, preferably 40 to 100C ~the boiling point when
the boiling point is below 100C) for 1 minute to 20 hours,
preferably 5 minutes to 2 hours. Then, the solvent is evaporated
at a pressure of 0.001 to 1 atm. and at a tempe~ature of 0C to
the boiling point of the solvent, preferably 40 to 80C (the
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boiling point ~hen the boiling point is below 80C) for ~ minutes
to 20 hours, prefcrably 10 minutes to 4 hours. If required,
the mixture is then dried at a pressure of 0.001 to 0.5 atms.
and at a temperature of 10 to 30C for 4 to 100 hours.
Pref-rably, water or both the auxiliary additive and
water, or an aqueous solution of the auxiliary additive, is added
to the water-soluble solid mentioned above (consisting of the
N4-acylcytosine arablnoside and the additive), and the mixture
is heated at 1 to 1.5 atms. and at 60 to 120C for 1 minute
to 4 hours to form a solution. When the auxiliary additive is
not added, the solution obtained be not allowed to cool
spontaneously, but rapidly cooled to -40C to +20C with flowing
water, ice water, dry ice-acetone, etc. This will increase
the solubllity of the solid further. Where the auxiliary
additive is added, the ingredients dissolve sufficiently without
this rapid cooling operation.
In order to prepare an aqueous solution of the N4-
acylcytosine arabinoside containing an organic solvent, an
aqueous solution of the N4-acylcytosine arabinoside not at all
containing the organic solvent is first prepared, and the
organic solvent is then added to the aqueous solution.
Alternatively, an aqueous solution containing the organic
solvent is added to the N4-acylcytosine arabinoside, and the
mixture is heated, followed by rapidly cooling the resulting
solution. Another alternative, which is sometimes difficult
when the amount of the organic solvent to be added is small,
is to add the organic solvent to the N4-acylcytosine arabinoside
and additive, heat the mixture, and add water to an aqueous
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solution of the auxiliary additive either dircctly or after
cooling the mi~ture to room temperature, to thereby form an
aqueous solution of the N4-acylcytosine arabinoside containing
the organic solvent. Another possible method is to add an
aqueous sol~tion of the additive or both the additive and the
auxiliary additive to an organic solvent containing the N4-
acylcytosine arabinoside. It would also be possible to add an
aqueous solution containing the additive, or both the additive
and the auxiliary additive, and an organic solvent to the N4-
acylcytosine arabinoside.
Examples of the solvent capable of dissolving the N4-
acylcytosine arabinoside and the additive include ketones such
as acetone and methyl ethyl ketone, esters such as ethyl acetate
and butyl acetate, aliphatic ethers such as diethyl ether and
methylethyl ether, cyclic ethers such as tetrahydTofuran,
tetrahydropyran and dioxane, amides such as dimethyl acetamide,
dimethyl formamide and diethyl acetamide, sulfoxides such as
dimethyl sulfoxide, alcohols such as methanol, ethanol, n-propanol
and isopropanol, bases such as pyridine and triethanolamines,
and acids such as formic acid and acetic acid. Of these,
the ketones, esters, cylic ethers, aliphatic ethers, amides
and sulfoxides are preferred because they do not decompose the
N4-acylcytosine arabinoside. The ketones, esters, aliphatic
ethers, cyclic monoethers and alcohols are preferred because
of their ease of evaporation. The ketones, amides and ethanol
are preferred because of their low toxicity. Accordingly, the
ketones are most preferred because of their superior stability
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and evaporability and low toxicity. Since the cyclic monoethers
are superior in stabi.lity, evaporability and solubility, they
are as preferred as the ketones if they are completely evaporated
after use. Since ethanol has good evaporability and solubility
and low toxicity, it is as preferred as the ketones if the time
of its contact with the N4-acylcytosine arabinoside is shortened.
It is time-consuming to evaporate the amides and the sulfoxides
completely, but otherwise, they are preferred in the next place
because of their superior stability and solubility and low
toxicity.
The composition of this invention can be formulated into
internal preparations (orally administrable preparations)
such as tablets, trouches, buccal agents, chewable preparations,
capsules, cachets, powders, granules, pills, extracts, fluid
extracts, solutions, elixirs, spirit-incorporated preparations,
syrups, lemonades, aromatic water preparations, emulsions or
suspensions by adding, if required, vehicles, binders, disintegrants,
coating agents, solvents, correctives, pH adjusters, thickening
agents, stabilizers, wetting agents, defoamers, lubricants,
colorants, color inhibitors, moisture-proof agents, flavoring
agents, etc. The composition can also be formulated into
externally applicable preparations such as ointments, creams,
pastes, liniments, plasters, ophthalmic ointments, suppositories
(through the anus, cavity or urethra), lotions, jellies, liquids
(eye drops, nasal drops, lavages and baths), dusts (spray and
powder), tablets, aerosols and the like by adding base agents,
thickening agents, defoamers, stabilizers, flavoring agents,
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colorants, solverlts> isotonicity-imparting agents, buffers or
pain-reducing or -eli~inatillg agents. If further required,
the composition can be formulated into injectables such as
water preparations or oil preparations by adding pain-reducing
or -eliminating agents, isotonicity-imparting agents, buffers,
extenders or solvents. Or it can be formed into inhalants in
aerosol form by adding solvents.
N4-Acylcytosine arabinosides having 14 to 24 carbon atoms
in the acyl group at the N4-position are especially preferred
because they exhibit high pharmacological actions in accordance
with the present invention. Those in which the acyl group is
a saturated or unsaturated and substituted or unsubstituted
acyl group selected from myristoyl, palmitoyl, margaroyl,
stearoyl, nonadecanoyl, arachidoyl, heneicosanoyl, behenoyl,
tricosanoyl, lignoceroyl, oleoyl, 5-methyl nonadecanoyl,
2-chlorostearoyl, 18-hydroxystearoyl and 2-mercaptostearoyl
exhibit favorable effects. Of these, N4-stearoylcytosine
arabinoside and N4-behenoylcytosine arabinoside show especially
favorable effects.
The N4-acylcytosine arabinosides having 14 to 24 carbon
atoms in the acyl group are quite insoluble in water (soluble
in a concentration of less than 0.00001% by weight), but become
soluble in water in an amount of 0.02 to 4% by weight by adding
0.1 to 20% by weight of the specific additive and preferably
also the same amount of the auxiliary additive.
Since the desirable concentration of the arabinoside
in injectables for clinical purposes is 0.01 to 1% by weight
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(preferably ad~inistered in a dosage of 0.1 to 1,000 mg,
preferably 1 to 100 ~ng, per day for an adult), an aqueous solution
of the arabinoside prepared in this invention has a sufficiently
high concentration for use as injectables.
All of the additives used to form the N4-acylcytosine
arabinoside compositions in this invention are known and commer-
cially available.
The N -acylcytosine arabinosides are obtained, for example,
by reacting cytosine arabinoside with acid anhydrides in the
presence o water. For example, N4-behenoylcytosine arabinoside
can be obtained in the following manner.
Cytosine arabinoside (1.23 millimoles) is dissolved in
2 ml of water, and 30 ml of dioxane and 2.47 millimoles of
behenic anhydride are further added. The mixture is heated to
80C to dissolve the precipitate. After stirring at 80C for
5 hours, the reaction mixture is allowed to cool, and the
precipitate is collected by filtration. The precipitate is
thoroughly washed with water, and dried. To the dried precipitate
is added n-hexane, and the mixture heated under reflux.
The product is cooled and collected by filtration. The product
is then washed with benzene and then with toluene.
Recrystallization from ethyl acetate affords N4-behenoylcytosine
arabinoside (AS-22) in a yield of 82.5%.
Other N4-acylcytosine arabinosides are synthesized by
using acid anhydrides corresponding to the acyl groups of the
desired products instead of behenic anhydride.
The following ~xamples illustrate the present invention
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in greater detail.
The determination of the pharmacological activity of
the compounds of this invention is performed using mice injected
with L-1210 leukemic cells. The L-1210 mouse leukemia is being
employed as a standard of judging anticancer activity both by
Cancer Institute, National Institute of Healthy of U. S. A. and
by Cancer Chemotherapy Center, Japanese Foundation for Cancer
Research on the ground that among perimental cancers, L-1210
leukemia on mice most predicts pharmacological effects of
drug against cancer on humans.
Specifically, the test is conducted as follows:
An N4-acylcytosine arabinoside is administered to groups of
mice each consisting of 10 mice of the CDFl line intraperitoneally
injected with 106, for each mouse, of L-1210 leukemic cells.
The average survival time (T) of the mice in the experimental
groups and the average survival time (C)of the mice in the
control groups are determined, and the pharmacological effect
of the N4-acylcytosine is expressed by the percentage of T based
on C (T/C ~).
The acute toxicity of the N4-acylcytosine arabinoside
is expressed by the dosage, per kilogram of body weight of mouse,
of the N4-acylcytosine arabinoside which causes five out of
10 male mice of the CDFl line as one group to die. This is
indicated as LD50 mg/kg.
The present invention will be described in more detail
by the following examples.
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Example 1
One gram of N4-behenoylcytosine arabinoside and each
of the additives shown in Table 1 in the amounts indicated
were added to 100 g of dioxane. The mixture was heated at 1
atm. and at 80C for 10 minutes, and the dioxane was evaporated
at 0.1 atm. and at 60C for 4 hours.
The residue and each of the auxiliary additives shown in
Table 2 (the numbers correspond to those in Table 1) in the
amounts indicated were added to 200 g of water. The mixture
was heated at 95C and at 1 atm. for 20 minutes with stirring,
and then çooled in ice water, followed by lyophilizing.
The compositions shown in Table 3 (the numbers also
correspond to those in Tables 1 and 2) were obtained as white
amorphous powders ~the composition Nos. 2 and 6 were white
semi-fluids) in the yields indicated.
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Table
No. Additive Weight (g)
1 HCO-10 [polyoxyethylene(10) hydrogenated 0.1
castor oil]
2 HCO-10 20
3 HCO-60 [polyoxyethylene(6o) hydrogenated
castor oil] 0.
4 HCO-60 5
HCO-60 20
6 HCO-200 [polyoxyethylene (200) hydrogenated
castor oil] 0.1
7 HCO-200 20
8 Polyoxyethylene ~60)-glycerin 0.1
trihydroxycaprate
9 Polyoxyethylene ~60)-glycerin
trihydroxycaprate 20
Polyoxyethylene ~60)-glycerin
dihydroxyarachidate 0.1
11 Polyoxyethylene ~60)-glycerin
dihydroxyarachidate 20
12 Polyoxyethylene ~60).glycerin
trihydroxystearate/glycerin
tristearate mixture (3:1) 0.1
13 Polyoxyethylene (60)-glycerine
trihydroxystearate/glycerine
tristearate mixture ~3:1) 20
14 HCO-60/sodium deoxycholate ~1:1) 0.2
lS HCO-60/sodium deoxycholate (70:1) 7.1
16 HCO-60/sodium d.eoxycholate (35:1) 7.2
; 17 HCO-60/potassium deoxycholate (25:1) 5.2
18 Deoxycholic acid 0.1
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lahle 1 (cont_n ed)
No. Additive Weight ~g~
19 Deoxycholic acid 20
Sodium deoxycholate 0.1
21 Sodium deoxycholate 20
22 Potassium deoxycholate 0.1
23 Potassium deoxycholate 20
24 Dehydrocholic acid 0.1
Dehydrocholic acid 20
26 Sodium cholate 0.1
27 Sodium cholate 20
28 Sodium lithocholate 0.1
29 Sodium lithocholate 20
Chenodeoxycholic acid 0.1
31 Chenodeoxycholic acid 20
32 Ursodeoxycholic acid 0.1
33 Ursodeoxycholic acid 20
34 Sodium hyodeoxycholate 0.1
Sodium hyodeoxycholate 20
36 Sodium lagodeoxycholate 0.1
37 Sodium lagodeoxycholate 20
38 Sodium hyocholate 0.1
39 Sodium hyocholate 20
Sodium phocaecholate 0.1
41 Sodium phocaecholate 20
42 Ammonium deoxycholate 0.1
43 Ammonium deoxycholate 20
44 Trimethylammonium deoxycholate 0.1
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Tablc 1 (continucd)_
No. Additive Weight ~g)
Trimethylammonium deoxycholate 20
46 Procaine deoxycholate 0.1
47 Procaine deoxycholate 20
48 HCO-60 5
49 HCO-60 S
HCO-60 S
51 HCO-60 5
52 HCO-60 5
53 HCO-60 5
54 HCO-60 5
HCO-60/sodium deoxycholate ~35:1) 7.2
56 HCO-60/sodium deoxycholate (35:1) 7.2
57 HCO-60/sodium deoxycholate (35:1) 7.2
58 HCO-60/sodium deoxycholate (35:1) 7.2
59 HCO-60/sodium deoxycholate (35:1) 7.2
HCO-60/sodium deoxycholate (35:1) 7.2
61 HCO-60/sodium deoxycholate (35:1) 7.2
62 HCO-60/sodium deoxycholate (35:1) 7.2
63 HCO-60/sodium deoxycholate (35:1) 7.2
64 HCO-60/sodium deoxycholate (35:1) 7.2
HCO-60/sodium deoxycholate (35:1) 7.2
66 HCO-60/sodium dehydrocholate (35:1) 7.2
67 HCO-60/sodium dehydrocholate (35:1) 7.2
68 HCO-60/sodium dehydrocholate (35:1) 7.2
69 HCO-60/sodium dehydrocholate (35:1) 7.2
HCO-60/sodium dehydrocholate (35:1) 7.2
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Ta)lc 1 (continucd)
No. Additive Weight
71 HCO-60/sodium dehydrocholate (35:1) 7.2
72 HCO-60/sodium dehydrocholate (35:1) 7.2
73 HCO-60/sodium dehydrocholate (35:1) 7.2
74 HCO-~0/sodium dehydrocholate (35:1) 7.2
HCO-60/sodium dehydrocholate (35:1) 7.2
76 HCO-60/sodium dehydrocholate (35:1) 7.2
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Table 2
No. Auxiliary ~dditive Weight (g~ -
1 Glycerol 20
2 Glycerol 0.1
3 Glucose 5
4 Glucose . 5
Glucose 5
6 Propylene glycol 20
7 Propylene glycol 0.1
8 Ribose 20
9 Ribose 0.1
Arabinose 20
11 Arabinose . 0.1
12 Fructose 20
13 Fructose 0.1
14 Sorbitol 20
Sorbitol 0.1
16 Mannitol 20
17 Mannitol 0.1
18 Lactose 20
19 Lactose 0.1
Saccharose 20
21 Saccharose 0.1
22 NaCl . 20
23 NaCl 0.1
24 KCl 20
- - 20 -
' ";
, . ,.: .
,
,
1063937
Table 2 rcontinucd)
No. Auxiliary . clitive _ Weight ~g~
KCl 0.1
26 CaC12 20
27 CaCl~ 0.1
28 MgC12 . 20
29 MgC12 0.1
Benzoic acid 20
31 Benzoic acid - 0.1
32 Sodium benzoate 20
33 Sodium benzoate 0.1
34 Potassium benzoate 20
Potassium benzoate 0.1
36 Salicylic acid 20
37 Salicyclic acid 0.1
38 Sodium salicylate 20
39 Sodium salicylate 0.1
Potassium salicylate 20
41 Potassium salicylate 0.1
42 Mixture of glucose and CaC12(10:1) 5.5
43 Mixture of glucose and MgC12(10:1) 5.5
44 Mixture of glucose and sodium benzoate (50:1) 5.1
Mixture of glucose and sodium salicylate (50:1) 5.1
46 Mixture of NaCl and CaC12 (1:1)
47 Mixture of NaCl and MgC12 (1:1)
48 Mixture of glucose and CaC12 (10:1) 5.5
49 Mixture of glucose and MgC12 (10:1) 5.5
Mixture of glucose and sodium salicylate (50:1) 5.1
- 21 -
~, .
1063937
Tablc 2_ (con_i ued) _
No. AuYiliary Acklitivc .~eight ~g~
51 CaC12
52 MgC12
53 NaCl
54 KCl
Mixture of glucose and CaC12 ~10:1) 5.5
56 Mixture of glucose and MgC12 ~10:1) 5.5
57 Mixture of gl~cose and sodium benzoate (50:1) 5.1
58 Mixture of glucose and sodium salicylate (50:1) 5.1
59 Glucose 5
NaCl
61 CaC12
62 MgC12
63 Sodium salicylate 0.1
64 Fructose 5
Lactose 5
66 Mixture of glucose and CaC12 (10:1) 5.5
67 Mixture of glucose and MgC12 ~10:1) 5.5
68 Mixture of glucose and sodium benzoate ~50:1) 5.1
69 Mixture of glucose and sodium salicylate ~50:1) 5.1
Glucose 5
71 NaCl
72 CaC12
73 MgC12
74 Sodium salicylate 0.1
Fructose 5
76 Lactose 5
1063937
Table 3
Yield
N Composition %
1 Be-C:HCO-lO:Gly(1:01:20) 98
2 Be-C:HC0-lO:Gly (1:20:0.1) 98
3 Be-C:HCO-60:Glu (1:0.1:5) 98
4 Be-C:HCO-60:Glu (1 : 5 : 5) 98
Be-C:HCO-60:Glu (1:20:5) 98
6 Be-C:HCO-200:Pro (1:0.1:20) 98
7 Be-C:HCO-200:Pro (1:20:0.1) 98
8 Be-C:Cap-60:Rib(1:0.1:20) 97
9 Be-C:Cap-60:Rib (1:20:0.1) 97
Be-C:Ara-60:Ara(1:0.1:20) 97
11 Be-C:Ara-60:Ara(1:20:0.1) 97
12 Be-C:Ste-60:Fru (1:0.1:20) 97
13 Be-C:Ste-60:Fru(1:20:0.1) 97
14 Be-C:HCO-60:DeoxNa:Sor(1:0.1:0.1:20) 97
Be-C:HCO-60:DeoxNa:Sor(1:7:0.1:0.1) 97
16 Be-C:HCO-60:DeoxNa:Man(1:7:0,2:20) 97
17 Be-C:HCO-60:DeoxNa:Man(1:7:0.2:0.1) 97
18 Be-C:DeoxH:Lac (l:G.1:20) 97
19 Be-C:DeoxH:Lac (1:20:0.1) 97
Be-C:DeoxNa:Sac(1:0.1:20) 97
21 Be-C:DeoxNa:Sac(1:20:0.1) 97
22 Be-C:DeoxK:NaCl(1:0.1:20) 97
- 23 -
~063937
Table 3 (continued)
No. Composition Yield
23 Be-C:DeoxK:NaCl (1:20:0.1) 97
24 Be-C:DehH:KCl (1:0.1:20) 97
Be-C:DehH:KCl (1;20:0.1) 97
26 Be-C:CNa:CaCl2(1:0.1:20) 97
27 Be-C:CNa:CaC12(1:20:0.1) 97
28 Be-C:I,iNa:MgC12(1:0.1:20) 97
29 Be-C:LiNa:MgCl2(1:20:0.1) 97
Be-C:CheH:BenH(1:0.1:20) 98
31 Be-C:CheH:BenH(1:20:0.1) 98
32 Be-C:UruH:BenNa(1:0.1:20) 98
33 Be-C:UruH:BenNa(1:20:0.1) 98
34 Be-C:HyodNa:BenK(1:0.1:20) 98
Be-C:HyodNa:BenK(1:20:0.1) 98
36 Be-C:LagodNa:SalH(1:0.1:20) 98
37 Be-C:La~odNa:SalH(1:20:0.1) 98
38 Be-C:HyoNa:SalNa(1:0.1:20) 98
39 Be-C:HyoNa:SalNa(1:20:0.1) 98
Be-C:PhoNa:SalK (1:0.1:20) 98
41 Be-C:PhoNa:SalK (1:20:0.1) 98
42 Be-C:DeoxNH4:Glu:CaCi2 (1:0.1:5:0.5) 97
43 Be-C:DeoxNH4:Glu:MgC12 (1:20:5:0.5) 97
44 Be-C:DeoxT:Glu:BenNa (1:0.1:5:0.1) 98
Be-C:DeoxT:Glu:SalNa (1:20:5:0.1) 98
46 Be-C:DeoxPr:NaCl:CaC12 (1:0.1:0.5:0.5)97
- 24 -
1063937
Table 3 (continue~)
No. Composition Yield
47 Be-C:DeoxPr:NaCl:~lgC12(1:20:0.5:0.5) 97
48 Be-C:HCO-60:Glu:CaC12(1:5:5:0.5) 97
49 Be-C:HCO-60:Glu:~lgC12(1:5:5:0.5) 97
Be-C:HCO-60:Glu:SalNa(1:5:5:0.1) 98
51 Be-C:HCO-60:CaC12 (1:5:1) 97
52 Be-C:HCO-60:MgC12 (1:5:1) 97
53 Be-C:HCO-60:NaCl (1:5:1) 97
54 Be-C:HCO-60:KCl (1:5:1) 97
Be-C:HCO-60:DeoxNa:Glu:CaC12
(1:7:0.2:5:0.5) 97
56 Be-C:HCO-60:DeoxNa:Glu:MgC12
(1:7:0.2:5:0.5) 97
57 Be-C:HCO-60:DeoxNa:Glu:BenNa
(1:7:0.2:5:0.1) 98
58 Be-C:HCO-60:DeoxNa:Glu:SalNa
(1:7:0.2:5~0.1) 98
59 Be-C:HCO-60:DeoxNa:Glu (1:7:0.2:5) 97
Be-C:HCO-60:DeoxNa:NaCl(1:7:0.2:1) 97
61 Be-C:HCO-60:DeoxNa:CaC12(1:7:0.2:1) 97
62 Be-C:HCO-60:DeoxNa:MgC12(1:7:0.2:1) 97
63 Be-C:HCO-60:DeoxNa:SalNa(1:7:0.2:0.1) 98
64 Be-C:HCO-60:DeoxNa:Fru(1:7:0.2:5) 97
Be-C:HCO-60:DeoxNa:Lac(1:7:0.2:5) 97
66 Be-C:HCO-60:DehNa:Glu:CaC12
(1:7:0.2:5:0.5) 97
67 Be-C:HCO-60:DehNa:Glu:MgC12
(1:7:0.2:5:0.5) 97
- 25 -
,
. , - .
,
1063937
Table 3 (continued)
No. Composition Yield
%
68 Be-C:HCO-60:DehNa:Clu:BenNa
(1:7:0.2:5:0.1) 98
~ 69 Be-C:HCO-60:DehNa:Glu:SalNa
; (1:7:0.2:5:0.1) 98
Be-C:HCO-60:DehNa:Glu(1:7:0.2:5)97
71 Be-C:HCO-60:DehNa:NaCl (1:7:0.2:1) 97
72 Be-C:HCO-60:DehNa:CaC12(1:7:0.2:1) 97
73 Be-C:HCO-60:DehNa:MgC12(1:7:0.2:1) 97
74 Be-C:HCO-60:DehNa:SalNa(1:7:0.2:0.1) 98
Be-C:HCO-60:DehNa:Fru (1:7:0.2:5) 97
76 Be-C:HCO-60:DehNa:Lac (1:7:0.2:5) 97
- 26 -
1063937
The abbreviations used in Tables 1, 2 and 3 and else-
where have the following meanings.
Be-C: N4-Behenoylcytosine arabinoside
HCO-10: Hydrogenated castor oil having bonded thereto
polyoxyethylene with an average degree of
polymerization of 10
HC0-60: Hydrogenated castor oil having bonded thereto
polyoxyethylene with an average degree of
polymerization of 60
HC0-200: Hydrogenated castor oil having bonded thereto
polyoxyethylene with an average degree of
polymerization of 200
Cap-60: Glycerin trihydroxycaprate having bonded thereto
polyoxyethylene with an average degree of
polymerization of 60
: Ara-60: Glycerin dihydroxyarachidate having bonded
thereto polyoxyethylene with an average degree
of polymerization of 60
Ste-60: Glycerin trihydroxystearate having bonded
thereto polyoxyethylene with an average degree
of polymerization of 60
DeoxNa: Sodium deoxycholate
DeoxH: Deoxycholic acid
DeoxK: Potassium deoxycholate
DehH: Dehydrocholic acid
DehNa: Sodium dehydrocholate
CNa: Sodium cholate
- 27 -
.
1063937
LiNa: Sodium lithocholate
CheH: Chenodeoxycholic acid
UruH: Ursodeoxycholic acid
HyodNa: Sodium hyodeoxycholate
LagodNa: Sodium lagodeoxycholate
HyoNa: Sodium hyocholate
PhoNa: Sodium Phocaecholate
DeoxNH4: Ammonium deoxycholate
DeoxT: Trimethylammonium deoxycholate
DeoxPr: Procaine deoxycholate
Gly: Glycerin
Glu: Glucose
Pro: Propylene glycol
Rib: Ribose
Ara: Arabinose
Fru: Fructose
Sor: Sorbitol
Man: Mannitol
Lac: Lactose
Sac: . Saccharose
BenH: Benzoic acid
BenNa: Sodium benzoate
Benk: Potassium benzoate
SalH: Salicylic acid
SalNa: Sodium salicylate
Salk: Potassium salicylate
1063937
In or~er to examine the anticancer activity of the
compositions of this invention and N4-behenoylcytosine arabino-
side alone in intravenous injection, groups of mice each con-
sisting of 10 male mice of the CDFl line injected with 106, per
mouse, of L-1210 leukemic cells were intravenously injected
with an aqueous solution containing 100 mg, calculated as the
N4-behenoylcytosine-arabinoside, of each of the compositions
or 100 mg of N4-behenoylcytosine arabinoside alone once on the
2nd and 6th days. The average survival time of the mice (T)
was determined, and divided by the average survival time (C)
of control groups not administered with the drugs at all to
obtain T/C % as shown in Table 4 below.
The toxicity of each of the compositions of the invention
and N4-behenoylcytosine arabinoside in intravenous injection
was expressed by the dosage, per kilogram of body weight of each
mouse, of the N4-behenoylcytosine arabinoside contained in each
composition which killed five out of 10 male mice of the
CDFl line as one group to obtain LDso mg/kg as shown in Table 4.
Table 4
Composition No. Anticancer Activity Toxicity
( T/C %)(LD50 mg/kg)
1 300 400
2 300 400
3 380 400
4 380 400
380 400
6 300 400
- 29 -
.
,, , - ~ ,
1~)63937
Tabl~ 4rcontinued~
- Composition No. Anticancer Activity Toxic ~
T C (LD50 mg g)
7 300 400
8 300 400
9 300 400
300 400
11 ' 300 400
12 380 400
13 380 400
14 380 400
380 400
16 380 400
: 17 380 400
18 300 400
19 300 400
380 400
21 380 400
22 300 400
23 300 400
24 300 400
300 400
26 300 400
27 300 400
28 300 400
29 300 400
300 400
: 31 300 400
~; - 30 -
: ~., . ,., ,.,: .. :
1063937
Table 4 (continued)
Composition No. Anticancer Activity Toxicity
(T/C ~)(LD50 mg/kg~
32 300 400
33 300 400
34 3pO 400
300 400
36 300 400
37 300 400
38 300 400
39 300 400
300 400
41 . 300 400
42 300 400
43 300 400
44 300 400
300 400
46 300 400
47 300 400
48 380 400
49 380 400
380 400
51 380 400
52 380 400
53 380 400
54 380 400
SS 380 400
, . . .
:' ' " ~-:' .
.
.
~ . .
1063937
Table 4 (continued)
Composition No. Anticancer Activity Toxicity
(T/C %)(LD50 mg/ g)
56 380 400
57 380 400
58 380 400
59 380 400
' 380 400
61 380 400
62 380 400
63 380 400
64 380 400
380 400
66 380 400
67 380 400
68 380 400
69 380 400
380 400
71 380 400
72 380 400
73 - 380 400
74 380 400
380 400
76 380 400
N -Behenoyl*
CA 150 400
Note: * N4-Behenoylcytosine arabinoside suspended
in a 0.5 ~ aqueous solution of methyl
cellulose
- 32 -
1063937
It can be scen from Table 4 that while the anticancer
activity (T/C %) of N4-behenoylcytosine arabinoside alone in
intravenous injection was 150%, the activity of the composition
of this invention showed an increase to 300 to 380~. -
Example 2
One gram each of the N4-acylcytosine arabinosides shown
in Table 5 and 7 g of HC0-60 were added to 1 kg of acetone.
The mixture was heated at 55C and at 1 atm. for 10 minutes,
and then the acetone was evaporated from the solution by heating
at 40C and at 0.1 atm. for 4 hours.
The residue and 5 g of glucose were added to 500 g of water,
and the mixture was heated with stirring at 95C and at 1 atm.
for 20 minutes and then cooled in ice water. It was then
lyophilized. Thus, the compositions shown in Table 6 (the numbers
correspond to those in Table 5) were obtained as white amorphous
powders in the yields shown in Table 6.
The anticancer activity (T/C %) of each of the compositions
obtained and N4-acylcytosine arabinosides alone in a 0.5% aqueous
solution of methyl cellulose in intravenous injection (100 mg/kg
x two times), and their toxicity [LD50 (mg/kg)] were determined in
the same way as in Example 1, and the results are shown in Table 7.
- 33 -
, ' ' - '
.
,
1063937
Table 5
N N4-Acylcytosine arabinoside
77 N4-Nonadecanoylcytosine arabinoside
78 N4-Arachidoylcytosine arabinoside
79 N4-Heneicosanoylcytosine arabinoside
N4-Tricosanoylcytosine arabinoside
81 N4-Lignoceroylcytosine arabinoside
82 N4-(5-Methylnonadecanoyl)cytosine arabinoside
Table 6
No. Composition Yield
77 N4-Nonadecanoyl CA:HC0-60:Glu(1:7:5) 97
78 N4-Arachidoyl CA:HC0-60:Glu (1:7:5) 97
79 N4-Heneicosanoyl CA:HCO-60:Glu (1:7:5) 97
N4-Tricosanoyl CA:HCO-60:Glu (1:7:5) 97
81 N4-Lignoceroyl CA:HC0-60:Glu (1:7:5) 97
82 N4-(5-Methylnonadecanoyl)CA:HCO-60:Glu(1:7:5) 97
CA: Cytosine arabinoside
HC0-60 ~ Glu: The same as those defined in Example 1
- 34 -
~063937
Table 7
Composition I~O. T/C (%) LDso (mg/kg)
77 350 400
78 350 400
79 350 400
350 400
81 , 350 400
82 350 400
N4-Nonadecanoylcytosine arabinoside 150 400
N4-Arachidoylcytosine arabinoside 150 400
N4-Heneicosanoylcytosine arabinoside 150 400
N4-Tricosanoylcytosine arabinoside 150 400
N4-Lignoceroylcytosine arabinoside 120 400
N4-(5-Methylnonadecanoyl)cytosine
arabinoside 150 400
Example 3
One gram each of the N4-acylcytosine arabinosides
indicated in Table 8 and each of the additives shown in Table 9
in the amounts indicated were added to 200 g of ethanol. The
mixture was heated at 60C and at 1 atm. for 20 minutes, and
then the ethanol was evaporated by heating at 40C and at 0.1 atm.
for 4 hours.
The residue and each of the auxiliary additives shown
in Table 10 in the amounts indicated were added to 1 kg of water.
The mixture was heated at 95C and at 1 atm. for 20 minutes with
stirring, and cooled in ice water, followed by lyophilizing.
- 35 -
. .
,~
... ' ,
' . , ' ' :` '
1063937
Thus, the compositions shown in Table 11 ~the numbers correspond
to those in Tables 8 to 10) were obtained as white amorphous
powders in the yields shown in Table 11.
The anticancer activity (T/C %) and per oral ~oxicity
LDso (mg/kg) of these compositions were determined in the same
manner as in Example 1 except that an aqueous solution of 400 mg,
per kg of body weight of mouse, of each composition calculated
as the N4-acylcytosine arabinoside, or 400 mg on the same basis
of a 0.5% aqueous solution of methyl cellulose containing each
of the N4-acylcytosine arabinosides alone was administered
orally to the L-1210 leukemia-inoculated mice three times (on
the 3rd, 5th and 7th days). The results are shown in Table 12.
Table 8
No. N4-Acylcytosine arabinoside
.
83 N4-Stearoylcytosine arabinoside
84 N4-Stearoylcytosine arabinoside
N4-Stearoylcytosine arabinoside
86 N4-Stearoylcytosine arabinoside
87 N4-Myristoylcytosine arabinoside
88 N4-Palmitoylcytosine arabinoside
89 N4-Margaroylcytosine arabinoside
N4-(2-Chlorostearoyl)cytosine arabinoside
91 N -(18-Hydroxystearoyl)cytosine arabinoside
92 N -(2-Mercaptostearoyl)cytosine arabinoside
93 N4-Margaroylcytosine arabinoside
94 N4-Margaroylcytosine arabinoside
- 36 -
,
1063937
Table 8 (continued)
No. N4-Acylcytosine arabinoside
N4-Margaroylcytosine arabinoside
: 96 N4-Valeroylcytosine arabinoside
97 N4-Stearoylcytosine arabinoside
,
Table 9
No. Additive Weight (g)
-
83 HCO-60 5
84 HCO-60 5
HCO-60 5
86 Sodium deoxycholate 5
87 HC0-60/sodium deoxycholate (1:1) 4
88 HC0-60/sodium deoxycholate ~1:1) 4
89 HC0-60/sodium deoxycholate (1:1) 4
HC0-60/sodium deoxycholate (1:1) 4
91 HC0-60/sodium deoxycholate (1:1) 4
92 HC0-60/sodium deoxycholate (1:1) 4
93 Mys-45 5
94 TW-30 5
HC0-60 5
96 HC0-60 5
: .
97 HC0-60 5
- 37 -
, . ., .
.
" ,,
1063937
Table 10
No. Auxiliary additive Weight (g)
83 Glucose 5
84 Glucose and MgC12 (1:2) 15
Glucose and CaCl~ 2) 15
86 Glucose 5
87 Glucose , 5
88 Glucose 5
89 Glucose 5
Glucose S
91 ~lucose 5
92 Glucose 5
93 Glucose 5
94 Glucose 5
MgS04 10
96 Glucose 5
97 NaBr 0.1
- 38 -
1063937
Table 11
No. Composition Yield
83 St-CA:HCO-60:Glu (1:5:5) 97
84 St-CA:HCO-60:Glu:MgC12 (1:5:5:10) 97
St-CA:HCO-60:Glu:CaC12 (1:5:5:10) 97
86 St-CA:DeoxNa:Glu (1:5:5) 97
87 My-CA:HCO-60:DeoxNa:Glu (1:2:2:5) 97
88 Pa-CA:HCO-60:DeoxNa:Glu (1:2:2:5) 97
89 Ma-CA:HCO-60:DeoxNa:Glu (1:2:2:5) g7
2-Cl-St-CA:HCO-60:DeoxNa:Glu (1:2:2:5) 97
91 18-Hy-St-CA:HCO-60:DeoxNa:Glu(1:2:2:5) 97
92 2-Me-St-CA:HCO-60:DeoxyNa:Glu(1:2:2:5) 97
93 Ma-CA:Mys-45:Glu(1:5:5) 97
94 Ma-CA:TW-30:Glu (1:5:5) 97
Ma-CA:HCO-60:MgSO4 (1:5:10) 97
96 Va-CA:HCO-60:Glu (1:5:5) 97
97 St-CA:HCO-60:NaBr (1:5:0.1) 97
Compari-
son Va-CA
- 39 -
.Y'
1063937
Table 12
Composition No. T/C (%) LDso (mg/kg)
83 300 above 1600
84 380 above 1600
3B0 above 1600
86 350 above 1600
87 ' 350 1600
88 290 above 1600
89 290 above 1600
290 above 1600
91 290 above 1600
92 290 above 1600
93 250 1600
94 250 1600
- 95 350 above 1600
96 140 above 1600
97 280 above 1600
Comparisons
ST-CA 150 above 1600
My-CA 150 . 1600
Pa-CA 150 above 1600
Ma-CA 150 above 1600
2-Cl-St-CA 150 above 1600
18-Hy-St-CA 150 above 1600
2-Me-St-CA 150 above 1600
Va-CA 110 above 1600
- 40 -
1063937
The abbreviations used in Tables 9 to 12 have the
following meanings.
St-CA: N4-Stearoylcytosine arabinoside
My-CA: N4-Myristoylcytosine arabinoside
Pa-CA: N4-Palmitoylcytosine arabinoside
Ma-CA: N4-Margaroylcytosine arabinoside
2-Cl-St-CA: N4-(2-Chlorostearoyl)cytosine arabinoside
18-~ly-St-CA: N4-(18-Hydroxystearoyl)cytosine arabinoside
2-Me-St-CA: N4-(2-Mercaptostearoyl)cytosine arabinoside
Mys-45: Stearic acid ester of polyoxyethylene with an
average degree of polymerization of 45
TW-30: Lanolin havingbonded thereto polyoxyethylene
with an average degree of polymerization of 30
Va-CA: N4-Valeroylcytosine arabinoside
It can be seen from Table 12 that while the anticancer
activity (T/C %) in oral administration of each of the N4-
acylcytosine arabinosides alone is 150%, the activities of the
compositions containing the N4-acylcytosine arabinosides in-
crease to 300 - 380%.
Example 4
Instead of oral administration in Example 3, 400 mg,
per kilogram of body weight of mouse, of each of the composi-
tions in powder form (calculated as the N4-acylcytosine ara-
binoside) or of a mixture of each N4-acylcytosine arabinoside
and methyl cellulose (2:1) was solidified in a rocket form,
,
- 41 -
1063937
and administered through the anus three times (once each on the
3rd, 5th and 7th days). The anticancer activities (T/C ~) and
LDso values (mg/kg) in intra-anal administration were determined,
and the results are shown in Table 13.
From these results, it is seen that the results were
much the same for both oral and intra-anal administrations.
Table 13
Compound No. T/C (%) LDso (mg/kg)
83 300 above 1600
84 380 above 1600
380 above 1600
86 350 above 1600
87 350 1600
88 280 above 1600
89 280 above 1600
280 above 1600
91 280 above 1600
92 280 above 1600
93 250 1600
94 250 1600
350 above 1600
96 140 above 1600
97 300 above 1600
- 42,-
1063937
Table 13 (continued)
Compound ~o. TtC (~) LD~n (mg/kg)
Comparisons
St-CA 150 above 1600
My-CA 150 1600
Pa-CA 150 above 1600
Ma-CA 150 above 1600
2-Cl-St-CA . 150 above 1600
18-Hy-St-CA 150 above 1600
2-Me-St-CA 150 above 1600
Va-CA 110 above 1600
Example 5
One gram of N4-behenoylcytosine arabinoside (AS-22 for
brevity) and each of the additives shown in Table 14 in the
amounts indicated were added to 400 ml of acetone. The mix-
ture was heated at 55C and at 1 atm. for 10 minutes, and then
the acetone was evaporated from the solution by heating at 55C
and at 0.2 atm. for 2 hours. The residue was dried in a vacuum
desiccator at 20C and at 0.1 atm. for 20 hours. Thus, water-
soluble compositions containing AS-22 were obtained in yields
of 95 to 99%.
The solubility of each of the water-soluble solids of AS-22
was determined in the following manner. When a clear aqueous
solution can be obtained by adding 100 ml of a 0.9% aqueous
solution of sodium chloride to a water-soluble solid containing 1 g
of AS-22, stirring the mixture in a hot water bath at 90C and
rapidly cooling the resulting aqueous solution in an ice water bath
,
- 43 -
"
- ,
.., ' ~
1063937
the solubility of the water-soluble solid is determined to be
more than lg/100 ml. When a precipitate forms under the above
conditions, 100 ml. of a 0.9~ aqueous solution of sodium
chloride is further added, and the aforesaid heating-rapid
cooling procedure is repeated to form a solution. The solubility
in this case is calculated as the amount of AS-22 dissolved
per lO0 ml of water: The results are also shown in Table 14.
Table 14
No. Additive Weight solubility
(g) of AS-22
~g/100 ~1)
98 Polyoxyethylene (40) hydrogenated 1.8 0.5
castor oil 2 ~1
(HC0-40) 100 ~1
99 Polyoxyethylene (50) hydrogenated 1.8 0.5
castor oil 2 ~1
(HC0-50) 100 ~1
100 Polyoxyethylene (60) hydrogenated 1.8 0.5
castor oil 2 ~1
(HC0-60) 100 ~1
lOl Polyoxyethylene (80) hydrogenated 1.8 0.5
castor oil 2 ~1
(HCO-80) 100 ~1
102 Polyoxyethylene (100) hydrogenated 1.8 0-5
castor oil 2 71
(HCO-lO0) 100 ~1
103 Polyoxyethylene (40) castor oil 1.8 0.5
(C0-40TX) 2 ~1
100 ~1
104 Polyoxyethylene (60) castor oil 1.8 0.5
(CO-60TX) 2 71
100 71
- 44 -
~ s, , . ,.,.~ "~ ~
~063937
I-a_ c ]~ (eontinucd)
No Ad(Ijtive Weight Solubilit
o,~~~
(~/lOb ml)
105 Polyo~ycthylenc (30) lanolin 1.8 0.5
(TIY-30) 2 71
. 100 ~1
106 Polyo~ycthylenc (40) stearatc 1.5 0.5
t~IYS-40) 2 ~ 1
100 71
107 Polyoxycthylene (~5) stearate 1.5 0.5
~MYS-45) 2 71
lO0 71
108 Polyoxyethylene (5S) stearate 1.5 0.5
(~S-55) 2 ~ 1
100 ~1
- 109 Sodium desoxycholate 2 0.5
>1
''................................................... 100 ~1
`- 110 AS-22 alone is suspended without the 0 0
use of the additive
' - '
. . .
In the above table, the abbreviations in the p~rentheses
are the commodity numbers of the products (under the trademark
Nikkol) of Nikko Chemicals Co., Ltd. It is expected that
products of similar compositions made by other manufacturers
such as Nissan Chemical Co., Ltd. or Atlas Company will have
much the same solubilities.
. ' ' .
,' '
- 45 -
,,
~ '.
.
1063937
Examplc 6
To l ~ Or ~S-22 wcrc ad~cd 2 ~ of polyoxycthylcnc (60)
hydrogenatcd castor oil (Nikkol IIC0-60, a ploduct for use ;n
drugs by Nikko Chcn1icals Co., Itd.), O.l g of polyoxycthylene (40)
stcarate (Nikkol ~IYS-40), and 50 ml of cthanol. Thc m~xture was
hcated at 75C for 5 minutes; The e~hanol was evaporatcd at 55C
and at 0.2 atm. for 30 minutcs, and thc residue was dried at 20C
and at O.l atm. for 4 hours to afford about 3.l ~ of a water-
soluble solid. To the water-soluble solid containing AS-22 thus
obtained, lO0 ml of a 0.8% aqueous solution of sodium chloride
was added, and the mixture was heated in a hot water bath at 90C
to form a solution which was rapidly cooled in an ice water bath
to afford an almost colorless clear 1% aqueous solution of AS-22.
The aqueous solution was passed through a membrane filter (with
a pore size of 0.45 ~u) to give a 1% injectable of AS-22.
Example 7
.
To l g of AS-22 were added 2 g of polyoxyethylene (60)
~ .
hydrogenated castor oil (Nikkol ~IC0-60) and 50 ml of tetra-
hydrofuran, and the mixture was heated at 50C for lO minutes.
To the resulting solution was added 0.8 g of sodium chloride in
pcwder form, and then the tetrahydrofuran was evaporated at 50C
and at 0.2 atm. for 30 minutes. The residue was dried in a
desiccator at 20C and at O.l atm. for 20 hours to afford a
w;ter-soluble tacky white solid of AS-22.~1C0-60.NaCl. Water
(100 ml) was added to the white solid, and the mixture heated
in a hot water bath at 90C to form a solution which was rapidly
c~oled in an ice water bath to afford a 1% aqueous solution of
- 46 -
. ~ .
,. , , '
-
.;,. , .
.~ '
1063937
AS-22. The aqueous solution was filtered by means of a mil-
lipore filter of the type described in Example 6 to afford
a 1% aqueous injectable of AS-22 (containing 1% of AS-22,
2% of HC0-60, and 0.8~ of NaCl).
Example 8
Dimethyl acetamide t2 ml) was added to 1 g of AS-22 and
4 g of polyoxyethylene (60) hydrogenated castor oil (Nikkol
HC0-60), and the mixture was heated at 90C and at 1 atm. for
lO minutes to form a solution. When the solution was allowed
to cool to room temperature, it became a white solid. Water
(100 ml) was added to the solid, and the mixture was heated
at 90C for 2 hours with vigorous stirring to dissolve the solid.
The solution was dipped in ice water, and cooled with vigorous
stirring. Thus, a 1% aqueous solution of AS-22 was obtained.
Example 9
Dimethyl acetamide (2 ml) was added to 1 g of AS-22,
and the mixture heated at 90C and at 1 atm. for 10 minutes. To
the solution was added 100 ml of a 4% aqueous solution of poly-
oxyethylene (60) hydrogenated castor oil (Nikkol HC0-60) heated
to 90C. The mixture was stirred vigorously at 90C for 2 hours.
A part of AS-22 dissolved. The mixture was rapidly cooled with
ice water, and filtered to remove the insoluble part of AS-22 to
afford a 0.1% aqueous solution of AS-22.
- - 47 -
. . - , .
"' ' ''
1063937
Example 10
An aqueous solution containing 2~ of dimethyl acetamide
and ~ of polyoxyethylene (60) hydrogenated castor oil (Nikkol
~ICO-60) was added to 0.1 g of AS-22, and the mixture heated at
90C. A part of AS-22 dissolved. The mixture was rapidly
cooled with ice water, and tlien filtered to remove the insoluble
AS^22 to thereby aford a 0.02% aqueous solution of AS-22.
Example 11
Water (100 ml) was added to 0.3 g, 1.5 g or 3.0 g of
a water-soluble solid obtained in the same way as in Example 5
from 1 part of AS-22 and 2 parts of polyoxyethylene (60)
hydrogenated castor oil. Each of the mixtures obtained was
heated at 90C for 2 hours. The solution was allowed to cool
to room temperature.
On the other hand, 100 ml of water was added to 0.3 g,
1.5 g or 3.0 g of a water-soluble solid prepared from 1 part of
AS-22 and 2 parts of polyoxyethylene (60) hydrogenated castor oil,
and each of the mixtures was heated at 90C for 2 hours. The
solution was rapidly cooled in ice water.
It was found that by rapid cooling, the aqueous solutions
; containing 0.1 g, 0.5 g and 1.0 g of AS-22 were all clear, but
by spontaneous cooling, the aqueous solutions containing 0.1 g
and 0.5 g of AS-22 were clear but the aqueous solution containing
1.0 g of AS-22 was non-transparent showing that AS-22 did not
completely dissolve. Samples rapidly cooled were still trans-
parent after storage for a week at room temperature, whereas
samples spontaneously cooled developed white precipitates on
storage for only one day at room temperature.
:
- 48 -
~063937
Example 12
Ethanol (40 ml) was added to 1 g of AS-22 and 2 g of
polyoxyethylene (60) hydrogenated castor oil (Nikkol HCO-60),
and the mixture heated at 60C. The ethanol was evaporated from
the solution at 60C under reduced pressure. The residue was
dried in a vacuum desiccator to afford 3 g of a white water-
soluble solid. To the solid was added 100 ml of a 0.8% aqueous
solution of sodium chloride, and the mixture heated in a hot
water bath at 90C for 2 hours. The solution was rapidly cooled
in an ice bath, and filtered by a millipore filter having a pore
size of 0.45 ~u to remove microorganisms present. Thus, an in-
jectable of AS-22 containing 1% of AS-22, 2% of HCO-60 and 0.8%
of NaCl was obtained.
Example 13
Each of the organic solvents indicated in Table 15 was
added in the amounts indicated to 1 g of AS-22 and 2 g of poly-
oxyethylene (60) hydrogenated castor oil (Nikkol HCO-60).
Each of the mixtures was heated at each of the temperatures
indicated in Table 15 at 1 atm for 10 minutes. The solvent was-
evaporated from the resulting solution at each of the temperatures
shown in Table 15 at 0.2 atm. The residue was dried at 25C
for 20 hours in a vacuum desiccator. The amount of water required
to dissolve the resulting water-soluble solids was determined.
It was found that all of these solids dissolved in 50 ml of
water, and no difference was observed.
As a control, the same procedure as above was performed
using 100 ml of hexane incapable of dissolving AS-22. It was .
- 49 -
, . ', ' ' ~'':
. .
1063937
found that ad~ition of hexane to a mixture of AS-22 and poly-
oxyethylene (60) hydrogenated castor oil resulted in the dis-
solving of only the polyoxyethylene hydrogenated castor oil
in water, and AS-22 remained almost undissolved in water.
Table 15
Dissolving Evaporating Solubility
Solvent Amount temper- temperature of AS-22
(ml) ature(C) (C) (g/100 ml)
Methanol 200 75 50 2
Ethanol 50 75 50 2
n-Propanol 50 60 60 2
iso-Propanol 50 60 60 2
Tetrahydrofuran 20 50 50 2
Tetrahydropyran 20 50 50 2
Dioxane 50 80 80 2
Acetone 200 50 50 2
Methyl ethyl ketone 200 50 50 2
Dimethyl acetamide 20 80 80 2
Dimethyl formamide 20 80 80 2
Dimethyl sulfoxide 20 80 80 2
Diethyl acetamide20 80 -. 80 2
Ethyl acetate 500 50 40 2
Butyl acetate 500 50 40 2
Diethyl ether 500 35 - 40 2
Methylethyl ether500 35 40 2
Pyridine 50 40 40 2
Triethanolamine 50 40 40 2
Acetic acid 50 40 50 2
n-Hexane 1000 50 50 less than 0.02
- 50 -
1063937
~xample 14
Acetone (50 ml) was added to l g of AS-22 and 4 g of
polyoxyethylene (60) hydrogenated castor oil (Nikkol HCO-60)
and the mixture heated at 55C and at 1 atm. for 2 hours. They
did not completely dissolve. The acetone was evaporated from
the mixture at 55C and at 0.2 atm. for 2 hours, and then
water was added in an amount of 100 ml. The mixture was heated
at 90C, and the resulting solution was rapidly cooled in an ice
bath to obtain an aqueous solution containing 1% of AS-22 and
4% of HCO-60.
Example 15
One gram of each of the N4-acylcytosine arabinosides
shown in Table 16 and 10 g of polyoxyethylene (60) hydrogenated
castor oil (Nikkol HCO-60) were dissolved in 200 ml of acetone
by heating at 55C and at 1 atm. for 10 minutes. The acetone
was evaporated at 55C and at 0.2 atm. for 2 hours. The residue
was dried in a vacuum desiccator at 20C and at 0.1 atm. for
20 hours to obtain an aqueous solid containing each of the N4-
acylcytosine arabinosides in a yield of 99%. Water (100 ml)
was added to 1 g of each of the water-soluble solids consisting
of each of the N4-acylcytosine arabinosides and polyoxyethylene
(60) hardened castor oil in a ratio of 1:10, and the mixture
stirred in a hot water bath at 90C. The aqueous solution
obtained was rapidly cooled in an ice water bath. Thus, clear
solutions were obtained in a yield of 99%.
~o63937
Table 16
Compound Acyl group in the AS:I-ICO-60 Proportions
No. N4-acylcytosine ratio in of As and
arabinoside the water- HCO-60 in
soluble aqueous
solid solution
111 Valeryl 1 : 10 0.1~ 1%
112 Lauroyl 1 : 10 0.1
113 Palmitoyl 1 : 10 0.1
114 Stearoyl 1 : 10 0.1
115 Arachidoyl 1 : 10 0.1
116 Heneicosanoyl 1 : 10 0.1
117 Erucoyl 1 : 10 0.1
118 Behenoyl 1 : 10 0.1
119 Lignoceroyl 1 : 10 0.1
120 Hexatriacontanoyl 1 : 10 0.1
121 2-Chlorostearoyl 1 : 10 0.1
122 18-Hydroxystearoyl 1 : 10 0.1
123 2-Mercaptostearoyl 1 : 10 0.1
124 5-~ethylnonadecanoyl 1 : 10 0.1 - 1
125 Benzoyl 1 : 10 0.1
126 Phenylbutyryl 1 : 10 0.1
127 p-Nitrobenzoyl 1 : 10 0.1
* AS: N4-acylcytosine arabinoside
- 52 -
~o63937
Example 16
The solubility (I) in water of a composition consiting
of an N4-acylcytosine arabinoside, an additive and an auxiliary
additive (to be referred to as a three-component composition) J
the solubility (II) in an aqueous solution of an auxiliary
additive of a composition consisting of an N4-acylcytosine
arabinoside and an additive (to be referred to as a two-component
.
composition), and the solubility (III) in water of the two-
component composition were determined under the following three
conditons.
(A) When stirring is performed at room temperature
for 10 minutes;
(B) When stirring is performed at 90C for 10 minutes,
and then the stirred mixture is allowed to cool to
room temperature; and
~C) When stirring is performed at 90C for 10 minutes,
and then the stirred mixture is rapidly cooled in
ice water, and thereafter its temperature is retu~ned
to room temperature.
Specific procedures were as follows: One gram, calculated
as the N4-behenoylcytosine arabinoside (AS-22 for brevity), of
each of the three-component composition Nos. 1 to 76 obtained
in Example 1 was added to 100 ml of water, and the mixture was
stirred at room temperature for 10 minutes and then filtered by
a millipore filter (pore size 0.45~u). The aqueous solution was
distilled at 50C and at 0.02 atm. for 4 hours. The residue
was vacuum dried at 0.001 atm. and at 20C for 24 hours.
... .
1063937
By measuring the weight of the dried residue, the solubility of
the composition under this condition (IA) was obtained.
Similarly, the solubilities of the composition under conditions
B and C (IB and IC) were measured. The solubilities are
expressed by the weight in grams of AS^22 dissolved in 100 ml
of water. When AS-22 (1 g) completely dissolved, the above
composition was fur~her added to the resulting solution to see
whether AS-22 dissolved beyond 1 g under the same conditions.
Next, one gram, calculated as AS-22, of each of the
two-component composition Nos. 1' to 76' obtained in Example 1
by dioxane treatment (each consisting of 1 g of AS-22 and each
of the additives shown in Table 1 in the amounts shown in Table l;
the compositions 1' to 76' correspond to Nos. 1 to 76) was added
to an aqueous solution consisting of lOO ml of water and each
of the auxiliary additives shown in Table 2 in the amounts in-
dicated in Table 2. In the same manner as described above,
its solubilities under the conditions A, B and C (IIA, IIB and
IIC) were measured.
Finally, 1 g, calculated as AS-22, of each of the two-
component composition Nos. 1' to 76' was added to 100 ml of
water, and in the same manner as above, its solubilities under
the conditions A, B and C (IIIA, IIIB and IIIC) were measured.
The results are shown in Table 17.
- 54 -
1063937
Table 17
Composi- I C~posi- II III
tion ~ tion
No. A B ~ No. A B C A B C
: l0.1 0.1 0.1 1' 0.050.1 0.1 0.01 0.050.1
21 >1 >1 2' 0.3 ~1 ~1 0.01 0.3 ~1
30.1 0.1 0.1 3' 0.050.1 0.1 0.01 0.050.1
41 >1 ~1 4' 0.4 >1 >1 0.01 0.4 ?1
S1 >1 ~1 5' 0.4 ~1 ~1 0.01 0.4 ~1
60.1 0.1 0.1 6' 0.050.1 0.1 0.01 0.050.1
71 >1 ?1 7' 0.3 >1 >1 0.01 0.3 ?1
81 0.1 0.1 8' 0.050.1 0.1 0.01 0.050.1
91 >1 ~1 9' 0.3 >1 ~1 0.01 0.3 ~1
: 100.1 0.1 0.1 10' 0.050.1 0.1 0.01 0.050.1
111 >1 ~1 11' 0.3 >1 >1 0.01 0.3 71
120.1 0.1 0.1 12' 0.050.1 0.1 0.01 0.050.1
131 >1 ~1 13' 0.3 ~1 ~1 0.01 0.3 ~1
140.2 0.2 0.2 14' 0.1 0.2 0.2 0.01 0.1 0.2
151 >1 ~1 15' 0.5 >1 ~1 0.01 0.5 ~1
161 ~1 ~1 16' 0.5 >1 ~1 0.01 0.5 ~ 1
171 ~1 ~1 17' 0.5 ~ 1 ~1 0.01 0.5 ~1
180.1 0.1 0.1 18' 0.050.1 0.1 0.01 0.050.1
19 -1 >1 >1 19' 0.3 ~1 ?1 0.01 0.3 ~1
200.1 0.1 0.1 20' 0.050.1 0.1 0.01 0.050.1
211 >1 71 21' 0.3 ~1 ~1 0.01 0.3 ~1
220.1 0.1 0.1 22' 0.050.1 0.1 0.01 0.050.1
231 ~1 ~1 23' 0.3 ?1 ~1 0.01 0.3 ~ 1
240.1 0.1 0.1 24' 0.050.1 0.1 0.01 0.050.1
- 55 -
i,' ' '
.. . .
1063937
Tablc 17 (continu~d)
Compos i - I Compos i - I I I I I
tion tion
No. A B C No. A B C A B C
1 71 ~1 25 '0.3 ~ 1 710.01 0.3 71
26 0.1 0.1 0.1 26'0.05 0.1 0.1 0.01 0.05 0.1
27 1 ~1 ~1 27' 0.3 ~1 ~10.01 0.3 7 1
28 0.1 0.1 0.1 28'0.05 0.1 0.1 0.01 0.05 0.1
29 1 ~1 ~1. 29' 0.3 71 ~10.01~0.3 ~1
0.1 0.1 0.1 30'0.05 0.1 0.1 0.01 0.05 0.1
31 1 ~1 >1 31' 0.3 ~1 ~10.01 0.3 71
32 0.1 0.1 0.1 32'0.05 0.1 0.1 0.01 0.05 0.1
33 1 ~1 >1 33' 0.3 71 ~10.01 0.3 ~1
34 0.1 0.1 0.1 34'0.05 0.1 0.1 0.01 0.05 0.1
1 ~1 ~1 35' 0.3 ~1 ~10.01 0.3 ~1
36 0.1 0.1 0.1 36'0.05 0.1 0.1 0.01 O.OS 0.1
37 1 ~1 ~1 37 '0.3 ~1 ~10.01 0.3 ~ 1
38 0.1 0.1 0.1 38'0.05 0.1 0.1 0.01 0.05 0.1
39 1 ~1 ~1 39' 0.3 ~1 ~10.01 0.3 ~1
0.1 0.1 0.1 40'0.05 0.1 0.1 0.01 0.05 0.1
41 1 >1 ~1 41' 0.3 ~1 ~10.01 0.3 ~1
42 0.1 0.1 0.1 42'0.05 0.1 0.1 0.01 0.05 0.1
43 1 ~1 ~1 43' 0.3 71 ~10.01 0.3 ~1
44 0.1 0.1 0.1 44'0.05 0.1 0.1 0.01 0.05 0.1
1 >1 >1 45' 0.3 ~1 ~10.01 0.3 ~1
46 0.1 0.1 0.1 46'0.05 0.1 0.1 0.01 0.05 0.1
47 1 ~1 ~1 47' 0.3 ~1 >10.01 0.3 71
48 1 71 ~1 48' 0.4 ~1 ~10.01 0.4 ~1
49 1 ~1 ~1 49 ' 0.4 ,71 71 0.01 0.4>1
1 >1 >1 50' 0.4 ~1 ~1 0.01 0.471
- 56 -
' '
., : . .
~o63937
Table 17 (continued)
Composi- I Composi- II III
tlon A B C No. A B C A B C
51 1 ~l ~1 51' 0.4 >1 ~1 0.010.4 ~1
- 52 1 >1 >1 52' 0.4 >1 >1 0.010-.4 ~1
53 1 ~1 >1 53' 0.4 ~1 ~1 0.01~-4 >1
54 1 ?1 71 54t 0.4 ~1 71 0.010.4 ~1
1 ~1 ~1 55' 0.5 ~ 1 >1 0.010l5 ~1
56 1 >1 ~1 56' 0.5 ~ 1 >1 0.019.5 ~1
57 1 >1 ~1 57' 0.5 ~1 ~1 0.010.5 ~1
58 1 71 ~1 58' 0.5 71 ~1 0.010.5 71
59 1 ~1 ~l 59' 0.5 71 ~1 0.010.5 >1
1 71 ~1 60' 0.5 ~1 71 0.010.5 ~1
61 1 ~1 >1 61' 0.5 ~1 ~1 0.010.5 ~1
62 1 71 71 62' 0.5 ~1 71 0.010.5 ~1
63 1 ~1 >1 63' 0.5 ~1 71 0.010.5 ~1
- 64 1 ~1 >1 64' 0.5 ~1 ~l 0.010.5 71
1 ~1 ~1 65' 0.5 71 ~1 0.010.5 ~1
66 1 ~1 ~1 66' 0.5 71 71 0.010.5 ~1
67 1 ~1 ~1 67' 0.5 ~1 71 0.010.5 ~1
68 1 ~1 ~1 68' 0.5 71 >1 0.010.5 ~ 1
69 1 71 ~1 69' 0.5 ~ 1 ~71 0.010.5 71
1 ~1 ~1 70' 0.5 71 ~1 0.010.5 71
71 1 ~1 ~1 71' 0.5 ~1 ~1 0.010.5 7 1
72 1 71 ~1 72' 0.5 71 71 0.010.5 ~71
73 1 ~1 71 73' 0.5 ~1 ~1 0.010.5 71
74 1 71 ~1 74' 0.5 71 ~ 1 0.010.5 71
1 71 ~1 75' 0.5 71 ~1 0.010.5 ~1
76 1 ~1 ~1 76' 0.5 71 ;7 1 0.010.5 7 1
~,.
- 57 -
, ' ~ ,
~063937
It can be seen from Table 17 that under condition C
in which after stirring for 10 minutes at 90C, the stirred
mixturc has rapidly cooled in ice water and then its temperature
was returned to room temperature, compositions containing the
auxiliary additives and those not containing them had the same
solubilities, but that under condition B in which after stirring
for 10 minutes at 90C, the stirred mixture was allowed to cool
spontaneously to roo'm temperature, compositions containing the
auxiliary additives had higher solubility than those not contain-
ing the auxiliary additives.
On the other hand, under condition A in which stirring
was effected for 10 minutes at room temperature, the three-
component compositions which contained the auxiliary additives
from the start had higher solubility than the two-component
compositions to which the auxiliary additives were added later
in the form of aqueous solution.
While the invention has been described in detail and
with referencè to the specific embodiments thereof, it will
be apparent to one skilled in the art that various changes and
modifications can be made therein without departing from the
spirir and scope thereof.
:'
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