Composition for activating function of phagocytes

A pharmaceutical or veterinary composition for activating the function of phygocytes comprising as an effective ingredient a certain tocopheryl glycoside. The composition is useful for treating infectious diseases and malignant tumor.

FIELD OF THE INVENTION 
The present invention relates to a composition for activating the function 
of phagocytes. More particularly, it relates to a pharmaceutical or 
veterinary composition which contains as an active ingredient a certain 
tocopheryl glycoside and is useful for treating bacterial or viral 
infections in human and other mammals by activating the function of 
phagocytes. A method for activating the function of phagocytes is also 
included in the present invention. 
BACKGROUND OF THE INVENTION 
Phagocytes such as neutrophils, etc. play an important role in biophylaxis 
by phagocytosis, digestion and detoxication of foreign substances from the 
outside. Recently, it has been found that, in a host suffering from 
bacterial or viral infections, lowering of the function of phagocytes, 
lymphocytes and the like is observed and, when the function of them is 
lowered, such a host is liable to get infections and infections are liable 
to become severe. From this point of view, various studies relating to 
activation of the function of phagocytes and lymphocytes have been made 
and some drugs useful for activating the function of lymphocytes have been 
already developed and used in the treatment of and infectious diseases. 
However, for activating the function of phagocytes, only a few compounds, 
such as levamisole, have been known. 
On the other hand, during the study of pharmacological activities of 
tocopheryl glycosides, the present inventors have surprisingly found that 
certain tocopheryl glycosides are useful for augmenting migration and 
phagocytosis of phagocytes such as neutrophils and thereby the function of 
phagocytes are activated. 
OBJECTS AND SUMMARY OF THE INVENTION 
One object of the present invention is to provide a composition for 
activating the function of phagocytes. 
Another object of the present invention is to provide a method for 
activating the function of phagocytes. 
These objects as well as other objects and advantages of the present 
invention will become apparent to those skilled in the art from the 
following description. 
According to the present invention, there is provided a composition for 
activating the function of phagocytes which comprises a compound of the 
formula: 
##STR1## 
wherein R.sub.1 is a glucose or mannose residue; R.sub.2 and R.sub.3 are, 
the same or different, hydrogen or methyl, and a pharmaceutically or 
veterinary acceptable carrier or diluent. The compound of the formula [I] 
is composed of tocopherol and a sugar which are commonly used in drugs and 
food and, therefore, toxicity of the compound is very low. The composition 
for activating the function of phagocytes of the present invention is 
useful in treatment of infectious diseases by administrating it to human 
or other mammals orally, parenterally or externally. Thus, the method for 
activating the function of phagocytes of the present invention comprises 
orally or parenterally administering or topically applying the compound of 
the formula [I], preferably, in the form of the above composition, to a 
human subject or other mammals requiring activation of the function of 
phagocytes. 
DETAILED DESCRIPTION OF THE INVENTION 
In the formula [I], the group --C.sub.16 H.sub.33 at 2-position in the 
3,4-dihydrobenzopyran ring represents 4,8,12-trimethyltridecyl group of 
the side chain at the 2-position of tocopherol. Further, although there 
exist isomers in the sugar introduced into the hydroxy group at 
6-position, all the isomers including the mixture of the isomers and the 
isolated isomer are fallen within the scope of formula [I]. The compound 
of the formula [I] wherein R.sub.1 is glucose residue has been already 
known in the prior art (The 103rd Annula Meeting of The Pharmaceutical 
Society of Japan, Apr. 4-6, 1983). Further, the compound of the formula 
[I] is disclosed in the co-pending Japanese Patent Application No. 
151895/1984 assigned to the present assignee as an antiallergic agent. 
However, it is believed that the compound of the formula [I] has not been 
used heretofore in the prior art for activating the function of 
phagocytes. 
In the present invention, preferably, both R.sub.2 and R.sub.3 in the 
formula [I] are methyl. That is, the preferred compound of the formula [I] 
is .sub..alpha. -tocopheryl glycoside. The representative examples of the 
compound of the formula [I] are dl-.alpha.-tocopheryl glucoside and 
dl-.alpha.-tocopheryl mannoside. 
The compound of the formula [I] is prepared according to the method 
described in the above Japanese Patent Application No. 151895/1984. 
That is, tocopherol is reacted with an appropriate peracetylated sugar in 
an appropriate solvent at an elevated temperature, for example 80.degree. 
to 100.degree. C., for an appropriate period of time, for example, 3 to 7 
hours. By this reaction, the corresponding acetylated sugar residue is 
introduced at the 6-position to give an acetylated derivative which is an 
intermediate for the preparation of the compound of the formula [I]. The 
reaction proceeds preferably by using ethylene glycol diacetate or 
nitrobenzene as a solvent and adding p-toluenesulfonic acid as a catalyst. 
Tocopherol used as the starting material may be any one of .alpha.-, 
.beta.-, .gamma.- and .delta.-tocopherols. The peracetylated sugar is 
known or is prepared by acetylating the desired sugar according to a 
conventional acetylation method. 
The compound of the formula [I] can be prepared by deacetylating the 
acetylated derivative thus obtained according to a known method, for 
example, by heating it under reflux in absolute methanol in the presence 
of sodium methoxide and then treating with an ion-exchange regimen such as 
Amberlite IR-120 (H.sup.+ type). The compound of the formula [I] is 
obtained as a stable crystalline material which is soluble in an organic 
solvent such as alcohol, chloroform, benzene, etc. and insoluble in water 
and can be further purified by a conventional method such as 
recystallization, etc. 
The compound of the formula [I] thus obtained has very low toxicity. For 
example, both dl-.alpha.-tocopheryl glucoside and dl-.alpha.-tocopheryl 
mannoside show LD.sub.50 values in rat of .gtoreq.5000 mg/kg (p.o.) and 
.gtoreq.500 mg/kg (i.p.). 
Thus, the composition for activating the function of phagocytes of the 
present invention can be prepared in the dosage form suitable for oral, 
parenteral or external administration such as tablets, powders, granules, 
syrups, injectable preparations, eye lotions, ointments, creams, 
emulsions, aqueous alcoholic solutions and the like by incorporating a 
non-toxic but effective amount of the compound of the formula [I] with one 
or more pharmaceutically or veterinary acceptable carriers or diluents 
such as binders, disintegrators, lubricants, solvents, agents for making 
isotonic, emulsifiers, suspending agents, stabilizers and the like 
according to a conventional pharmaceutical or veterinary technique. 
Preferably, the composition of the present invention is formulated in a 
dosage unit form containing 1 to 100 mg of the compound of the formula 
[I]. 
The composition for activating the function of phagocytes of the present 
invention can be administered to human subjects or other mammals orally, 
parenterally or externally for treating infectious diseases. For example, 
in case of treating human subjects, the composition is very useful for the 
treatment of infectious diseases caused by abnormality of the function of 
phagocytes such as Chediak-Higashi syndrome, lazy leukocyte syndrome, 
chronic granulomatosis and juvenile periodontitis. 
The method for activating the function of phagocytes of the present 
invention is carried out by orally or parenterally administering or 
topically applying the compound of the formula [I], preferably, in the 
form of the above composition, to a human subject or other mammals 
requiring activation of the function of phagocytes. The dosage can be 
appropriately chosen according to conditions to be treated, the route of 
administration. However, usually, it is preferable to administer 1 to 100 
mg/kg of the compound of the formula [I] per a single dose with daily 
dosage regimen of 100 to 1000 mg/kg of the compound of the formula [I].

The following reference examples and examples further illustrate the 
present invention in detail but are not to be construed to limit the scope 
thereof. 
REFERENCE EAMPLE 1 
6-O-(.beta.-2,3,4,6-Tetraacetylmannopyranocyl)-dl-.alpha.-tocopherol 
dl-.alpha.-Tocopherol (10 g, 23.25 mmol) and .beta.-D-mannopyranose 
pentaacetate (3.3 g, 8.46 mmol) were dissolved in nitrobenzene (5 ml) and 
p-toluenesulfonic acid (75 mg, 0.44 mmol) was added to the solution. The 
reaction system was displaced with nitrogen and was reacted in an oil bath 
at 90.degree. C. under reduced pressure of 20 mmHg. 
The progress of the reaction was followed by thin layer chromatography 
(developing solvent: benzene-ethyl acetate (10:1)). After 5 hours, benzene 
(100 ml) was added to the reaction mixture and the mixture was washed with 
water (3.times.100 ml) and a saturated aqueous solution of sodium chloride 
(3.times.100 ml). The benzene layer was dried with anhydrous sodium 
sulfate and evaporated under reduced pressure to give a dark brown oily 
product (13 g). 
This material (13 g) was chromatographed on a column of silica gel (550 g) 
and eluted with benzene-ethyl acetate (9:1) to give the title product (2.4 
g, 38%) as a yellow oil. 
TLC [benzene-ethyl acetate (10:1)]: Rf=0.3 (single spot). 
IR .nu..sub.max.sup.KBr : 1756 (C.dbd.O) cm.sup.-1. 
MS: m/Z 760 (M.sup.+). 
REFERENCE EXAMPLE 2 
dl-.alpha.-Tocopherylmannoside 
The product (2.4 g, 3.12 mmol) of Reference Example 1 was dissolved in dry 
methanol (8 ml) and 0.1N sodium methoxide (2 ml) was added to the 
solution. The mixture was heated under reflux in a water bath. After 5 
minutes, the reaction mixture was cooled and neutralized with Amberlite 
IR-120 (H.sup.+ type). After decolorization with charcoal, the reaction 
mixture was filtered and the filtrate was evaporated under reduced 
pressure to give the title compound (1.23 g, 68%) as white crystals. 
The crystals thus obtained were further recrystallized from acetone to give 
the further purified title compound (0.95 g, 19%). 
m.p. 128.degree.-130.degree. C. 
TLC [chloroform-methanol (5:1)]: Rf=0.3. 
IR .nu..sub.max.sup.KBr : 3390, 1160 (OH; sugar) cm.sup.-1. 
MS: m/Z 592 (M.sup.+). 
REFERENCE EXAMPLE 3 
6-O-(.beta.-2,3,4,6-Tetraacetylmannopyranocyl)-d-.delta.-tocopherol 
d-.delta.-Tocopherol (10 g, 25 mmol) and .beta.-D-mannopyranose 
pentaacetate (3.3 g, 8.86 mmol) were dissolved in nitrobenzene (5 ml) and 
p-toluenesulfonic acid (80 mg, 0.5 mmol) was added to the solution. 
The reaction system was displaced with nitrogen and was reacted in an oil 
bath at 80.degree. C. under reduced pressure of 20 mmHg. After 4 hours, 
benzene (100 ml) was added to the reaction mixture and the mixture was 
washed with water (3.times.100 ml) and a saturated aqueous solution of 
sodium chloride (3.times.100 ml). 
The benzene layer was dried with anhydrous sodium sulfate and evaporated 
under reduced pressure to give a dark brown oily product (20 g). 
This material (20 g) was chromatographed on a column of silica gel (550 g) 
and eluted with benzene-ethyl acetate to give the title product (3.8 g, 
61%) as a yellow oil. 
TLC [benzene-ethyl acetate (10:1)]: Rf=0.3 (single spot). 
IR .nu..sub.max.sup.KBr : 1760 (C.dbd.O) cm.sup.-1. 
MS: m/Z 732 (M.sup.+). 
REFERENCE EXAMPLE 4 
d-.delta.-Tocopherylmannoside 
The product (3.8 g, 5.2 mmol) of Example 3 was dissolved in dry methanol (8 
ml) and 0.1N sodium methoxide (2 ml) was added to the solution. 
After 5 minutes, the reaction mixture was neutralized with Amberlite IR-120 
(H.sup.+ type) and decolorized with charcoal. The mixture was filtered and 
the filtrate was evaporated under reduced pressure to give (2.1 g, 72%) of 
the title product as a white crystals. 
The crystals obtained were recrystallized from acetone to give the purified 
title compound (1.36 g, 30%). 
m.p. 187.degree.-189.degree. C. 
TLC [chloroform-methanol (5:1)] Rf=0.3 (single spot). 
IR .nu..sub.max.sup.KBr : 3390, 1160 (OH; sugar) cm.sup.-1. 
MS: m/Z 564 (M.sup.+). 
REFERENCE EXAMPLE 5 
According to the same procedure as described in the above Reference 
Examples, the following compounds of formula [I] were prepared by using 
the corresponding tocopherol and the peracethyl sugar through the 
corresponding acetylated derivative. 
dl-.alpha.-tocopherylglucoside (m.p. 140.degree.-141.degree. C.). 
d-.delta.-tocopherylglucoside (m.p. 46.degree.-49.degree. C.). 
EXAMPLE 1 
______________________________________ 
Ingredients Parts by weight 
______________________________________ 
dl-.alpha.-Tocopherylglucoside 
30 
Calcium phosphate 490 
Crystalline cellulose 
350 
Carboxymethyl cellulose 
120 
Magnesium stearate 
10 
______________________________________ 
These ingredients were thoroughly mixed and directly tabletted to give 
tablets for oral administration. 
______________________________________ 
Ingredients Parts by weight 
______________________________________ 
dl-.alpha.-Tocopherylglucoside 
380 
Lactose 480 
Polyvinyl pyrrolidone 
45 
Hydroxypropylcellulose 
95 
______________________________________ 
EXAMPLE 2 
According to a standard wet granulation technique, granules for oral 
administration was prepared from these ingredients. 
EXAMPLE 3 
______________________________________ 
Ingredients Parts by weight 
______________________________________ 
dl-.alpha.-Tocopherylmannoside 
5 
Distilled water for injection 
950 
______________________________________ 
The ingredients were admixed to prepare a solution and the solution was 
sterilized by filtration to obtain an injectable preparation. 
EXAMPLE 4 
______________________________________ 
Ingredients Parts by weight 
______________________________________ 
d-.delta.-Tocopherylglucoside 
20 
Bees wax 100 
Paraffin wax 60 
Lanolin 30 
Isopropyl myristate 60 
Squalane 80 
Liquid paraffin 250 
Polyoxyethylenesorbitan monostearate 
18 
Propylene glycol 50 
Borax 7 
Water 325 
______________________________________ 
According to a standard method, an ointment was prepared from these 
ingredients. 
EXAMPLE 5 
______________________________________ 
Ingredients Parts by weight 
______________________________________ 
d-.delta.-Tocopherylmannoside 
50 
Stearic acid 20 
Cetanol 5 
Lanolin 20 
Isopropyl myristate 
20 
Squalane 30 
Liquid paraffin 80 
Polyoxyethylenecetyl ether 
17 
Triethanolamine 10 
Glycerin 40 
Flavor and Preservative 
q.p. 
Water up to 1000 parts 
______________________________________ 
According to a standard method, an emulsion preparation was prepared from 
these ingredients. 
The effect of tocopheryl glycosides on activation of the function of 
phagocytes was tested. The results are described hereinafter. 
(1) The effect on migration of neutrophils 
Neutrophils were separated from human peripheral blood and the effect of 
various tocopherylglycosides on random migration and chemotaxis of the 
neutrophils were measured by using Boyden chambers. 
Tocopherol or a tocopheryl glycoside dissolved in 1% dimethylsulfoxide was 
added to a suspension containing 2.5.times.10.sup.6 cells/ml of 
neutrophils so that a concentration of tocopherol or the tocopheryl 
glycoside was 1 .mu.g/ml. After 15 minutes incubation at 37.degree. C., 
the mixture was added to the upper part of the chamber. To the lower part 
of the chamber was added PBS in case of measuring random migration, or 
added 2.times.10.sup.-8 M of N-formyl-methionyl-leucyl-phenylalanine in 
case of measuring chemotaxis. Then, the chamber was incubated at 
37.degree. C. for 1 to 3 hours. After incubation, the filter was fixed and 
stained with hematoxylin, and then the number of neutrophils migrated to 
the bottom of the filter was counted by using a microscope. As a control, 
the same procedure was repeated except that tocopherol or the tocopheryl 
glycoside was not added. The results are shown in Table 1. In Table 1, the 
relative percentage of the counting obtained by addition of tocopherol or 
the tocopheryl glycoside is shown by taking the counting of the control as 
100%. 
TABLE 1 
______________________________________ 
random motoricity 
chemotaxis 
Sample (%) (%) 
______________________________________ 
Control 100 100 
dl-.alpha.-Tocopherol 
82 .+-. 12 94 .+-. 5 
dl-.alpha.-Tocopherylglucoside 
98 .+-. 34 140 .+-. 6 
dl-.alpha.-Tocopherylgalactoside 
64 .+-. 8 85 .+-. 3 
dl-.alpha.-Tocopherylmannoside 
71 .+-. 16 168 .+-. 3 
______________________________________ 
As is seen from Table 1, chemotaxis of neutrophils is specifically improved 
only by the tocopheryl glucoside having glucose or mannose residue at the 
6-position of 3,4-dihydrobenzopyran ring of tocopherol, while random 
migration thereof is scarecely effected. 
(2) The effect on phagocytosis of neutrophils 
Neutrophils were separated from human peripheral blood and a suspension 
containing 2.5.times.10.sup.6 cells/ml of neutrophils was prepared. 
Tocopherol or a different tocopheryl glycoside was added to 900 .mu.l of 
this suspension so that the concentration of tocopherol or the tocopheryl 
glycoside was 1 .mu.g/ml and the mixture was incubated at 37.degree. C. 
for 15 minutes. 
The suspension (100 .mu.l) containing 1.times.10.sup.9 cells/ml of 
opsonized Staphylococcus aureus (IFO 13276) labeled with fluorescein 
isothiocyanate was added to the mixture. The resulting mixture was 
incubated at 37.degree. C. for 15 minutes. Then, the number of cells 
incorporated into 100 cells of neutrophils was counted by using a 
fluorescence microscope. As a control, the same procedure was repeated 
except that tocopherol or the tocopheryl glycoside was not added. The 
results are shown in Table 2. In Table 2, the relative percentage of the 
counting obtained by addition of tocopherol or the tocopheryl glycoside is 
shown by taking the counting of the control as 100%. 
TABLE 2 
______________________________________ 
sample phagocytosis (%) 
______________________________________ 
Control 100 
dl-.alpha.-Tocopherol 
164 .+-. 16 
dl-.alpha.-Tocopherylglucoside 
168 .+-. 31 
dl-.alpha.-Tocopherylgalactoside 
112 .+-. 9 
dl-.alpha.-Tocopherylmannoside 
219 .+-. 21 
______________________________________ 
As is seen from Table 2, the tocopheryl glycoside of the formula [I] also 
remarkably activates phagocytosis of neutrophils. 
(3) In vivo test to Pseudomonas aeruginosa 
An aqueous injectable solution of dl-.alpha.-tocopheryl glucoside or 
dl-.alpha.-tocopheryl mannoside (see Example 3) was intravenously injected 
to ICR mice (body weight: 20 to 25 g, 10 mice per a group (5 female mice 
and 5 male mice)) once a day (dose: 1 mg/kg) for three days continuously. 
After the final injection, 2.5.times.10.sup.7 cells of Pseudomonas 
aeruginosa (P-1 strain, Ikaken) were injected i.p. and the percentage of 
survival animuls was determined after 24 hours. As a control, the same 
procedure was repeated except that an equal amount of distilled water was 
used instead of the tocopheryl glycoside and the percentage of survival 
animals was determined. As the result, the percentage of survival of the 
control group was 0%, whereas that of the group injected with 
dl-.alpha.-tocopherylglucoside or dl-.alpha.-tocopherylmannoside was 20%, 
respectively. 
From these results, it is clear that the tocoperyl glyucoside of formula 
[I] has an excellent activity for activating the function of phagocytes.