Method for treatment of allergic disorders and immune complex diseases

Therapeutic agent for the treatment of allergic disorders, immune complex diseases and tumors, which contains a human urinary acid protease as an active ingredient, and a method for treating allergic disorders, immune complex diseases and tumors by administering the said acid protease, which has never been used as a therapeutic agent for allergic disorders, immune complex diseases and tumors. Since the acid protease is a protein of human origin, the probability of adverse reactions such as anaphylactic shock due to the antigenicity of the acid protease is believed to be extremely small.

BACKGROUND OF THE INVENTION 
Allergic disorders are induced by antigen-antibody reactions. When an 
individual has been immunologically primed or sensitized, further contact 
with antigen can lead not only to secondary boosting of the immune 
response but can also cause tissue-damaging reactions, i.e., allergic 
disorders. The mechanism of pathogenesis of allergic disorders is 
presently believed as follows: 
An individual produces antibodies after exposure to pathogenic antigen. 
Secondary antigen exposure causes antigen-antibody reaction and the formed 
antigen-antibody complexes deposit on the tissues, and chemical mediators 
are released from sensitized cells. Then these mediators and/or the 
deposited antigen-antibody complexes damage tissues. 
Pathogenic antigens are xenogenic antigens (inhaled allergen, food 
allergen, drugs and so on), allogenic antigens and autologus antigens 
which are denatured autologus components of tissues or organs, and act as 
foreign substances. 
So-called allergic disorders may be classified into four types; 
(1) Type I allergy (anaphylactic-type), in which the antigen reacts with a 
specific class of antibody bound to mast cells or circulating basophils 
through a specialized region of the antibody. This leads to degranulation 
of the cells and release of vasoactive mediators; 
(2) Type II allergy (cytotoxic-type), in which the antibodies on the cell 
surface bind to an antigen and cause several reactions such as opsonic or 
immune phagocytosis of the cell, and cell lysis by the action of the 
complement system; 
(3) Type III allergy (Arthus type; immune complex mediated), in which a 
complex is formed between antigen and humoral antibody and causes 
activation of the complement system, platelet aggregation, microthrombi 
formation, and so on; 
(4) Type IV allergy (cell-mediated or delayed-type), in which 
thymus-derived lymphocytes (T cells) with specific receptors are 
stimulated by antigens and release mediators. In case of tissue rejection, 
these lymphocytes transform to kill certain cells with the 
histocompatibility antigen of the graft. 
Among the allergic reactions, Types I, III and IV allergies participate in, 
for example, bronchial asthma and each of these reactions is considered 
independently, or in combination to cause these disorders. The mechanism 
of induction of allergic disorders is considered as follows: an antigen 
which enters an organism is treated by macrophages and the immunological 
information is transmitted to the T cell-B cell system. The B cells which 
have received the information produce anibody (IgE antibody is mainly 
produced in Type I allergy and IgG antibody in Type II or Type III 
allergy). IgE antibody binds to basophils in circulation or to mast cells 
in the tissues, thereby establishing the sensitized state. Hereafter, the 
same antigen which enters the sensitized organism binds with the antibody 
on these cells and releases chemical mediators, such as histamine, or slow 
reacting substances of anaphylaxis (SRS-A). The chemical mediators thus 
released induce allergic symptoms such as erythema, edema, or increase of 
glandular secretion caused by contraction of smooth muscles and increase 
of capillary permeability. On the other hand, IgG-antibody binds 
polymorpho-nuclear leukocytes to achieve sensitization, and SRS-A as a 
chemical mediator is thought to be secreted. 
Agents for treatment of allergic disorders can achieve their therapeutic 
purpose by inhibiting any step in the above-mentioned processes. For 
example, xanthine derivatives, .beta.-adrenergic stimulants 
(.beta.-stimulants) or corticosteroids are used for treatment of bronchial 
asthma. However, unfavorable adverse reactions have often been observed in 
these drugs. For example, palpitation and tachycardia are reported in 
patients receiving xanthine derivatives and .beta.-stimulants. 
Furthermore, corticosteroids cause adverse reactions such as peptic ulcer 
and complication of bacterial infection. Anti-histamine agents are not 
effective for bronchial asthma; these agents sometimes make the asthma 
even worse by making it difficult to expectorate tracheal secretions. 
Immune complex diseases, represented by rheumatoid arthritis, systemic 
lupus erythematosus (SLE) and lupus nephritis, as implied by the name, are 
diseases which are induced by complexes of antigens with antibodies, i.e., 
immune complexes, and are type III allergies. Although the mechanism of 
occurence of these diseases is complicated and has many points which are 
left unclear, it is generally believed to follow the course described 
below. 
When bacterial or viral infections damage tissues, antibodies are produced 
against newly formed autoantigens or virally infected cells to form immune 
complexes. Since these immune complexes activate the complement system and 
platelets, vasoactive substances such as histamine and serotonin are 
released and the permeability of the blood vessels is increased. Then, the 
immune complexes in circulation enter and deposit along the basement 
membrane of the vessel wall whose permeability has been increased. Where 
the immune complexes have deposited, polymorphonuclear leukocytes are 
gathered by the action of the leukocyte chemotactic factors which have 
been formed by the action of the complement to the deposited immune 
complexes. The polymorphonuclear leukocytes, reacting with the immune 
complexes, release various tissue-damaging substances such as cathepsins D 
and E, collagenase, elastase and permeability factors, and these 
substances eventually damage the tissue. In patients with immune complex 
diseases such as SLE, levels of the complement in the serum are generally 
low and aggravation of the disease conditions is closely correlated with 
the decrease of the complement levels. This decline is thought to be due 
to plentiful consumption of the complement at the site of the reaction 
between antigens and antibodies taking place such as in kidneys and blood 
vessels. Further, the immune complexes also are related to blood 
coagulation systems, and it is believed that the immune complexes cause 
serious symptoms through diverse mechanism for example by acceleration of 
fibrinoid deposition on the damaged tissues. 
Today, there are several kinds of agents for the treatment of immune 
complex diseases: immunosuppressive agents such as steroids which suppress 
activation of the immune system, anti-inflammatory agents which reduce 
local inflammations and pain, or anticoagulative agents and antiplatelet 
agents which serve to improve abnormalities of the 
coagulation-fibrinolysis system in the blood vessels. However, these 
agents are not satisfactorily effective and are associated with strong 
adverse reactions. Thus, development of a medicine which is safe and 
highly effective in the treatment of the diseases is strongly desired. 
Furthermore, many agents have been developed for the treatment of malignant 
tumors. 
The anti-tumor agents are classified roughly into two types. The first 
includes so called cytotoxic drugs which directly suppress the growth of 
tumors. The second includes the drugs which indirectly control the growth 
of tumors by activating the immunological protective functions of the 
host. However, the former do not exhibit sufficient selective cytotoxicity 
against the tumor cells and are toxic also against normal cells, whereby 
the total amount of the agent which can be used is considerably limited. 
On the other hand, the latter, i.e., immunopotentiators, are generally 
safely used, less frequently exhibiting unfavorable adverse reactions 
compared to the former. However, tumors are originated from the normal 
cells of the patient, so that the tumor may not sufficiently be recognized 
as a foreign substance. Therefore, some immunopotentiators have an 
essential problem that they do not elicit sufficient anti-tumor effect. 
SUMMARY OF THE INVENTION 
An object of this invention is to provide an agent for the treatment of 
allergic disorders, immune complex diseases and tumors, which contains as 
an active ingredient a human urinary acid protease. 
More specifically, the object of this invention is to provide a therapeutic 
agent for allergic disorders, immune complex diseases and tumors causing 
no adverse reactions. 
Another object of this invention is to provide a method for treating 
allergic disorders, immune complex diseases and tumors by using a human 
urinary acid protease.

DETAILED DESCRIPTION OF THE INVENTION 
Under the background mentioned above, the present inventors have 
intensively investigated for the purpose of developing an effective 
therapeutic agent for treatment of allergic disorders, immune complex 
diseases and tumors. As the result, the inventors have found the facts 
that a human urinary acid protease exhibits a strong anti-allergic effect, 
remarkably suppresses various immune complex diseases, and also shows an 
excellent anti-tumor effect. The present invention has been accomplished 
based upon the above findings. 
The acid protease which constitutes the active ingredient of the agents of 
this invention is a known enzyme (Mirsky et al: J. Clin. Invest. 27, 818, 
1948) which has never been used as a therapeutic agent for allergic 
disorders, immune complex diseases and tumors. The acid protease can be 
obtained from human urine by suitably combining ordinary methods for 
isolating proteins such as salting out, adsorption chromatography on 
inorganic adsorbents, ion-exchange chromatography on an ion-exchange resin 
or gel chromatography using a molecular sieve. 
As a non-limiting example, human urine is passed through a DEAE-cellulose 
column equilibrated with 0.1 M acetate buffer solution (pH 5.3) by the 
method of Seijffers et al. (American Journal of Physiology, 206, 1106, 
1964) so as to have the acid protease adsorbed on the column. The protease 
is then eluted with the same buffer solution containing 0.3M sodium 
chloride. The eluate is concentrated, then further purified by gel 
chromatography using Sephadex G-100, and subjected to an acid treatment. 
Thus may be obtained the acid protease of this invention. The term "human 
urinary acid protease" is intended in this specification to mean the acid 
protease which may have the following physical and chemical properties and 
may be obtained from human urine. 
The acid protease obtained by the above method is found to possess a 
molecular weight of 32,000-38,000 by gel chromatography on Sephadex G-100. 
It has an isoelectric point in the range of pH 1 to 3 by 
isoelectric-focusing on Ampholine. It has a maximum absorption at 278 nm, 
shows a positive reaction to ninhydrin and is readily soluble in water and 
insoluble in ether and chloroform. The acid protease shows an excellent 
hydrolytic activity in an acidic range, pH value less than 7, when 
hemoglobin is used as a substrate. The acid protease is inhibited by 
pepstatin. The activity of the acid protease remains stable in an acidic 
range, pH value less than 7, while the acid protease loses its activity in 
alkaline range, pH value over 8. 
The following is an example of the procedure for isolation and purification 
of the acid protease in the present invention. However, as a matter of 
course, this example is merely illustrative of the procedure of isolation 
and purification of the acid protease and not intended to limit the method 
thereof. 
Example of the procedure for the isolation and purification: 
One hundred liter of human urine was concentrated to about one-thousandth 
of its initial volume using pressure ultrafiltration instrument 
(Pellicon.RTM., Millipore Co., and Diaflo.RTM., Amicon Co.) with a cut off 
of 10,000 daltons. One hundred mililiter of the concentrated urine was 
applied to a DEAE-cellulose (Whatman co.) column (2.5.times.20 cm ) 
equilibrated with 0.1M acetate buffer (pH 6.0) and eluted with the same 
buffer containing 0.3M sodium chloride. The eluate was concentrated about 
to 100 ml by ultrafiltration and subjected to dialysis. The dialyzed 
solution was then applied to a DEAE-Sepharose (Pharmacia Co.) column 
(2.5.times.20 cm ) under the same condition described above. The eluted 
fraction was concentrated to about 10 ml, and applied to a Sephadex G-100 
gel filtration column (2.5.times.90 cm) for further purification and 
simultaneous removal of pyrogens. About 80 ml of the solution thus 
obtained was adjusted to pH 2 with hydrochloric acid, followed by 
10-minute incubation at 37.degree. C. Then the solution was lyophilized to 
give about 20 mg of the acid protease. 
Now, the pharmacological action and toxicity of this acid protease will be 
described with reference to typical experiments below. The human urinary 
acid protease in this specification is not necessarily isolated and 
purified completely, and it can be used in the crude state so long as the 
impurities do not interrupt the pharmacological activities of the acid 
protease. 
EXPERIMENT 1 
Suppressive effect on production of anti-ovalbumin IgE antibody 
A group of 10 Wistar male rats weighing 180-200 g was used. 0.1 mg of 
ovalbumin and 20 mg of aluminum hydroxide gel were injected 
intraperitoneally. Starting from the next day, acid protease was injected 
intravenously once a day for 14 days. The sera were collected 7, 10 and 14 
days after the injection of ovalbumin and the anti-ovalbumin IgE antibody 
in the serum was determined by the homologus PCA reaction of the rats (H. 
Maruyama, et al., Folia Pharmacologica Japonica, 74, 179, 1978). The 
results are shown in FIG. 1. 
Production of anti-ovalbumin IgE antibody was significantly suppressed. 
EXPERIMENT 2 
Suppressive Effect on Bronchial Asthma 
A group of 10 Wistar male rats weighing 180-200 g was used. 0.1 mg of 
ovalbumin and 20 mg of aluminum hydroxide gel were injected 
intraperitoneally. Starting from the next day, acid protease was injected 
intravenously once a day for 14 days. On the 14th day 25 mg/kg of 
ovalbumin was injected intravenously to induce bronchial asthma. The 
tracheal contraction thus induced was measured by the method of Konzett 
and Rossler (Arch. Exptl. Path. Pharmacol. 195, 71, 1940). The contraction 
rate of the trachea was calculated regarding contraction of control group 
as 100. The results are shown in Table 1. 
TABLE 1 
______________________________________ 
contraction rate of 
trachea (%) 
______________________________________ 
Control 100 
Acid protease 
0.05 mg/kg 72 
0.5 mg/kg 47 
5 mg/kg 24 
______________________________________ 
The tracheal contraction was significantly suppressed by adminstration of 
the acid protease. 
EXPERIMENT 3 
Hydrolysis of Immune Complex 
Fifteen mg of a soluble immune complex [human IgG-rabbit anti-human IgG 
antibody] and 3 mg of the acid protease, trypsin, .alpha.-chymotrypsin or 
plasmin were dissolved in 1 ml of phosphate buffer solution (0.06M, pH 
6.0) and incubated at 37.degree. C. for 60 minutes. The reaction was 
stopped by the addition of 1 ml of 20% aqueous solution of perchloric 
acid. The supernatant was measured for absorbance at 280 nm to calculate 
the rate of the hydrolysis. The same procedure was repeated using human 
IgG instead of the immune complex. The ratio of the obtained hydrolysis 
rate of immune complex to that of human IgG was calculated. The results 
are shown in Table 2. 
Compared with the other proteases, the acid protease hydrolyzed the soluble 
immune complex more selectively than the normal human IgG. 
TABLE 2 
______________________________________ 
Relative rate 
of hydrolysis Hydrolysis ratio 
Immune (immune complex/ 
Human IgG 
complex human IgG) 
______________________________________ 
Acid protease 
100 380 3.8 
Trypsin 130 169 1.3 
.alpha.-Chymotrypsin 
22 26 1.2 
Plasmin 26 29 1.1 
______________________________________ 
EXPERIMENT 4 
Hydrolysis of Immune Complex 
Fifteen mg of a soluble immune complex [human IgG-rabbit anti-human IgG 
antibody] and 0.3 mg of the acid protease or trypsin were dissolved in 1 
ml of phosphate buffer solution (0.06M, pH 6.0) and 250 .mu.l of either 
rat serum or the same phosphate buffer solution was added to the mixture. 
Then the mixture was incubated at 37.degree. C. for 60 minutes. After 
completion of the reaction, the remaining human IgG was measured by the 
single radial immunodiffusion method using rabbit anti-human IgG serum so 
as to determine the ratio of the hydrolyzed immune complex to the 
initially incubated immune complex. The results are shown in Table 3. 
It was found that the hydrolytic activity of the acid protease on immune 
complex was not affected by the addition of serum. In contrast, the 
hydrolytic activity of trypsin was markedly reduced by the addition of 
serum. 
TABLE 3 
______________________________________ 
Ratio of hydrolysis (%) 
absence of serum 
Presence of serum 
______________________________________ 
Acid protease 
35 35 
Trypsin 19 2 
______________________________________ 
EXPERIMENT 5 
Suppressive Effect on Thyroiditis 
Suppressive effect of the acid protease on thyroiditis was investigated 
according to the method of Kotani et al. (Clinical Immunology, 9, 635, 
1977). A group of 10 BFU/HDK male rats (six weeks of age) was subjected to 
thymectomy and exposed to four repeated X-ray irradiations each of 200 
rads every two weeks. After 14 weeks following the thymectomy, these rats 
were sacrificed. The thyroid gland of each rat was removed and embedded in 
a paraffin block, then stained with hematoxylin-eosin or with azan, and 
examined for the degree of the infiltration of mononuclear cells, 
destruction of endoplasmic reticulum and glandular fibrosis so as to 
estimate the severity of the thyroiditis according to the grades of 0 to 
4. The acid protease was administered intravenously once a day. As a 
control, the acid protease was inactivated and administered. The results 
are shown in Table 4. 
Compared to the control, it was found that the acid protease decreased both 
occurrence and severity of thyroiditis in a dose dependent manner. 
TABLE 4 
______________________________________ 
Occurrence (%) 
Severity 
______________________________________ 
Control 90 3.4 .+-. 0.3 
Acid protease 
1 mg/kg 80 2.8 .+-. 0.4 
3 mg/kg 60 2.0 .+-. 0.1* 
10 mg/kg 40* 1.3 .+-. 0.1** 
______________________________________ 
*p &lt; 0.05, 
**p &lt; 0.01 
EXPERIMENT 6 
Suppressive Effect on Immune Complex Nephritis 
To a group of 10 C57BL male mice, 200 mg/kg of human IgG-rabbit anti-human 
IgG antibody complex was injected intravenously three times a day at an 
interval of 8 hours for three days. On the fourth day, the animals were 
sacrificed and the kidneys were removed. Deposition of the immune complex 
in the glomeruli was observed by the fluorescent antibody technique using 
goat anti-rabbit IgG antibody, labeled with fluorescein isothiocyanate. 
Further, the immune complex content in the serum was measured by the Clq 
binding assay. Urinary protein was measured with commercial test paper for 
the 34 mice from which urine had been collected before sacrifice. The acid 
protease was administered intravenously three times daily starting 
immediately after the injection of the immune complex. As a control, 
inactivated acid protease was given. The results are shown in Table 5. 
The immune complex content in the serum was decreased and the incidence of 
proteinuria and deposited immune complex in glomeruli were decreased by 
the administration of the acid protease. 
TABLE 5 
______________________________________ 
Deposition of 
Immune Proteinuria Immune complex 
complex (Number of on glomerulus 
content positive mice.sup.(a) / 
(Number of positive 
in serum total number 
mice/total number 
(.mu.g/ml) of mice tested) 
of mice used) 
______________________________________ 
Control 156 .+-. 18 
7/8 10/10 
Acid 
protease 
0.3 mg/kg 
128 .+-. 9 5/7 8/10 
1.0 mg/kg 
88 .+-. 12* 
4/10 5/10* 
3.0 mg/kg 
53 .+-. 7** 
3/9* 2/10** 
______________________________________ 
.sup.(a) Mice showing proteinuria grade 3 or 4 
*p &lt; 0.05, 
**p &lt; 0.01 
EXPERIMENT 7 
Suppressive Effect on Masugi Nephritis 
By the method of Suzuki et al. (Folia Pharmacologica Japonica, 68, 572, 
1972), rabbit anti-rat kidney serum was administered intravenously to a 
group of 10 Wistar male rats at a dose of 5 ml/kg. The acid protease was 
administered intravenously once a day after the administration of the 
anti-kidney serum. The immune complex content in the serum and protein 
content in the urine were measured weekly. As a control, inactivated acid 
protease was given. The results are shown in FIG. 2 and FIG. 3. 
In the group treated with the acid protease, a decrease in the protein 
content in urine and decrease in the immune complex content in serum were 
observed. 
EXPERIMENT 8 
Suppressive Effect on Spontaneous Lupus Nephritis in Mice 
The method of Abe et al. (The Ryumachi, 14, 43, 1974) was used. 
To a group of 16 16-week old female mice (NZB.times.NZW)F.sub.1, the acid 
protease was injected intravenously at a dose of 10 mg/kg once a day. As a 
control, inactivated acid protease was given. At intervals of 4 weeks, the 
protein content in the urine was tested with commercial test paper for 
grades from 0 to 4. The results are shown in FIG. 4. 
Six mice from each group were sacrificed at the age of 32 weeks for the 
observation of cell infiltration into the renal glomeruli. 
The administration of the acid protease was continued in the remaining mice 
from each group to determine the survival rate at the age of 50 weeks. The 
results are shown in Table 6. 
By the administration of the acid protease, increase in the urinary protein 
was significantly suppressed, the cell infiltration was decreased and the 
survival rate was increased. These results indicate that the acid protease 
suppresses the lupus nephritis in mice. 
TABLE 6 
______________________________________ 
Group treated with the 
Control group acid protease 
______________________________________ 
Cell Heavy infiltration 
Slight infiltration 
infiltration 
of small lymphocyte 
around the vessel wall 
and plasma cell 
Survival rate 
20% 80%* 
______________________________________ 
*p &lt; 0.05 
EXPERIMENT 9 
Suppressive Effect on Chronic Active Hepatitis 
In accordance with the method of Mayer et al. (British Journal of 
Experimental Pathology, 55, 498, 1974), human liver LSP (liver specific 
membrane lipoprotein) was repetitively injected together with Freund's 
complete adjuvant to a group of 10 rabbits to induce chronic active 
hepatitis. The acid protease was administered intravenously to the rabbits 
once a day for two weeks. Two weeks after the induction of the hepatitis, 
serum GOT and GPT activities were measured. As a control, inactivated acid 
protease was given. The results are shown in Table 7. 
The acid protease suppressed the increase of serum GOT, GPT activities in a 
dose-dependent manner. 
TABLE 7 
______________________________________ 
GOT activity 
GPT activity 
(unit/ml) (unit/ml) 
______________________________________ 
Control 337.5 .+-. 34.5 
395.1 .+-. 40.5 
Acid protease 
1 mg/kg 311.5 .+-. 27.4 
351.2 .+-. 37.8 
3 mg/kg 256.2 .+-. 41.1 
301.5 .+-. 29.7 
10 mg/kg 193.3 .+-. 36.3** 
245.7 .+-. 31.4** 
______________________________________ 
**p &lt; 0.01 
EXPERIMENT 10 
Suppressive Effect on Arthritis 
By the method of Tsukada et al. (The Ryumachi, 16, 255, 1976), 4 ml of a 
soluble bovine serum albumin (BSA)-rabbit anti-BSA antibody complex, 
containing 2 mg antibody-nitrogen, was injected into bilateral rabbit 
knee-joints (10 animals per group) once a day for 6 days to induce 
allergic arthritis. The acid protease was administered intraarticularly 
once a day from the first day of the administration of the BSA-anti-BSA 
antibody complex. Ten days after the first administration of the complex, 
the rabbits were sacrificed. The knee-joints were fixed with formalin, 
stained with hematoxylin-eosin and microscopically examined. As a control, 
inactivated acid protease was given. The results are shown in Table 8. 
In the control group, hyperplasia of synovial lining cells, pannus 
formation and cell infiltration of lymphoid follicles which are devoid of 
germinal centers were observed. In contrast, in the treated group, pannus 
formation and cell infiltration of lymphoid follicles were significantly 
decreased. 
TABLE 8 
______________________________________ 
Pathohistological observation 
Hyperplasia Cell infiltration 
of synovial 
Pannus of lymphoid 
lining cells 
formation 
follicles 
______________________________________ 
Control 10/10 9/10 8/10 
Acid protease 
1 mg/kg 10/10 5/10 4/10 
3 mg/kg 10/10 4/10* 2/10* 
10 mg/kg 10/10 3/10** 2/10* 
______________________________________ 
Expressed as number of positive animals/number of animals used 
*p &lt; 0.05 
**p &lt; 0.01 
EXPERIMENT 11 
Hydrolysis of Human Immune Complex 
Sera were collected from patients with rheumatoid arthritis, systemic lupus 
erythematosus (SLE) and hepatitis carrying immune complex. One ml portions 
of the sera were incubated with 10, 30 and 100 .mu.g of the acid protease 
at 37.degree. C. for 60 minutes. Then the immune complex content was 
determined by the hemolytic reaction of sheep red blood cells using guinea 
pig complement and taking human aggregated IgG as the standard, according 
to the method of Fust et al. (Atherosclerosis, 29, 181, 1978). 
Additionally, the sera from the patients with rheumatoid arthritis were 
assayed for rheumatoid factor (RA factor) by the hemagglutination reaction 
(RAHA test) by the method of Azuma et al. (The Ryumachi, 12, 330, 1972). 
The results are shown in Tables 9 and 10. 
The acid protease decreased the immune complex content in the serum of 
these patients in a dose dependent manner. The acid protease also 
decreased the RA factor content of the patients with rheumatoid arthritis. 
TABLE 9 
______________________________________ 
Hydrolysis of human immune complex 
Immune 
Serum Amount of acid complex con- 
Disease No. protease added (.mu.g/ml) 
tent (.mu.g/ml) 
______________________________________ 
Rheumatoid 
1 0 75 
arthritis 10 63 
30 52 
100 &lt;50 
2 0 234 
10 180 
30 151 
100 120 
3 0 103 
10 84 
30 63 
100 &lt;50 
Systemic 1 0 426 
lupus 10 384 
erythematosus 30 203 
100 150 
2 0 120 
10 74 
30 56 
100 &lt;50 
3 0 153 
10 108 
30 63 
100 50 
Hepatitis 1 0 63 
10 59 
30 &lt;50 
100 &lt;50 
2 0 72 
10 68 
30 59 
100 52 
______________________________________ 
TABLE 10 
______________________________________ 
Effect on RA factor 
Serum Amount of acid protease 
Maximum dilution to show 
No. added (.mu.g/ml) positive reaction by RAHA 
______________________________________ 
1 0 2048 
10 1024 
30 256 
100 32 
2 0 512 
10 128 
30 64 
100 64 
3 0 1024 
10 512 
30 128 
100 64 
4 0 256 
10 128 
30 64 
100 16 
______________________________________ 
EXPERIMENT 12 
Effect on the Growth of Cultured Human Breast Cancer Cells MX-1 and Mouse 
Leukemia Cells L1210 
Human breast cancer cells MX-1 and mouse leukemia cells L1210 were 
respectively suspended at a cell concentration of 10.sup.5 /ml in Eagle's 
medium containing 10% calf serum and test substances. The cells were 
cultured at 37.degree. C. under 5% CO.sub.2 for 48 hours. Then the number 
of viable cells was counted after staining with Tripan Blue. The growth 
inhibition rate was calculated according to the following equation and the 
results are shown in Table 11. 
##EQU1## 
TABLE 11 
______________________________________ 
Growth inhibition 
Concentration 
rate (%) 
added (.mu.g/ml) 
MX-1 L1210 
______________________________________ 
Acid protease 
30 15 8 
100 33 21 
300 55 30 
Mitomycin C 
100 47 62 
______________________________________ 
The acid protease inhibited the growth of tumor cells even at a low 
concentration. 
EXPERIMENT 13 
Effect on Human Breast Cancer Cell MX-1 Bearing Nude Mice 
A 2 mm-square piece of human breast cancer MX-1 was transplanted 
subcutaneously to a group of 5 nude mice (BALB/C, nu/nu). Two weeks after 
the transplantation, the acid protease was intravenously injected twice a 
day for 18 days. The tumor was weighed 32 days after the transplantation 
of the tumor. The results are shown in Table 12. 
TABLE 12 
______________________________________ 
Dose Weight of tumor 
(mg/kg) 
(g) 
______________________________________ 
Control 1.32 .+-. 0.09 
Acid protease 0.3 0.79 .+-. 0.2* 
3.0 0.65 .+-. 0.15* 
______________________________________ 
*p &lt; 0.05 
The acid protease exhibited significant antitumor effect. 
EXPERIMENT 14 
Effect on Leukemia Cells P388 Bearing Mice 
10.sup.5 of leukemia cells P388 were transplanted intraperitoneally to a 
group of 5 BDF.sub.1 male mice. 
The acid protease was injected intravenously into the mice twice a day 
beginning on the next day until the animals died. The average life span 
was caluculated and expressed as a percentage of control. The results are 
shown in Table 13. 
##EQU2## 
TABLE 13 
______________________________________ 
Dose Average life span 
(mg/kg) 
(%) 
______________________________________ 
Control 100 .+-. 5 
Acid protease 0.3 110 .+-. 5 
1.0 121 .+-. 9 
3.0 123 .+-. 9* 
Mitomycin C 0.5 136 .+-. 17 
______________________________________ 
*p &lt; 0.05 
The acid protease clearly increased average life span. 
EXPERIMENT 15 
Acute Toxicity 
The acid protease dissolved in physiological saline was administered 
intravenously or intraperitoneally to a group of 10 ddY male mice weighing 
20.+-.1 g at a dose of 2 g/kg. The mice were kept under daily observation 
for any toxicological symptoms for a week. No sign of any toxicity was 
observed throughout the period. 
As has been described in the above experiments, the acid protease which is 
the active ingredient of the pharmaceutical agent of the present invention 
suppressed production of IgE antibody and clearly exhibited a therapeutic 
effect on bronchial asthma. Furthermore, it clearly suppressed 
establishment and development of a number of diseases which are believed 
to be induced by immune complexes, for example, thyroiditis and nephritis. 
Moreover, the acid protease exhibited a strong antitumor effect. 
The amount of the acid protease required to obtain these effects is within 
a sufficiently safe range, according to the result of the acute toxicity 
study. Since the acid protease is a protein of human origin, the 
probability that it would induce serious adverse reactions, such as 
anaphylactic shock due to its antigenicity, is beleved to be extremely 
small. Therefore it is believed to constitute a highly useful therapeutic 
agent for allergic disorders such as bronchial asthma, urticaria, hay 
fever, contact dermatitis, food allergy, drug allergy, allergic rhinitis, 
hypersensitivity pneumonitis, various immune complex diseases such as 
systemic lupus erythematosus, glomerulonephritis with immune complex, 
periarteritis nodosa, rheumatoid arthritis, immune complex hepatitis, 
thyroiditis, serum sickness, myasthenia gravis, various tumors such as 
gastric cancer, lung cancer, liver cancer, colon cancer, breast cancer, 
prostatic cancer, uterine cancer, bladder cancer, leukemia, esophagal 
cancer, lymphomas. 
Although the agent of the present invention is generally prepared in the 
form of a solution for intravenous, subcutaneous, intramuscular or 
intraarticular injection, it may be used in the form of oral agent, 
inhalant or rectal suppository. Although the daily dose of the acid 
protease for an adult is in the range of from 1 to 1000 mg, preferably 
from 50 to 500 mg, it may be suitably increased or decreased depending on 
the symptom and the manner of application. 
Preparations for injection may include lyophilized preparations which are 
dissolved immediately before administration, as well as liquid 
preparations. Oral preparations may include capsules, tablets, granules, 
powders and liquid oral preparations. Inhalant may include a lyophilized 
preparation. For rectal administration, the form of suppository may be 
used conveniently. 
The acid protease of this invention can be formulated into agents by any of 
the conventional methods using pharmaceutically acceptable carriers or 
excipients. Example of solid carriers and excipients usable advantageously 
herein include common excipients such as lactose, mannitol, corn starch 
and potato starch, binders such as crystalline cellulose, cellulose 
derivatives, arabic gum, corn starch and gelatin; disintegrators such as 
corn starch, potato starch and calcium carbohydroxymethylcellulose; and 
lubricants such as talc and magnesium stearate. Examples of liquid 
carriers usable advantageously herein include distilled water for 
injection, physiological saline solution, vegetable oils for injection and 
glycols such as propylene glycol and polyethylene glycol. 
Now, typical but non-limiting fomulations of the agent of this invention 
will be shown below. 
FORMULATION 1 
In 10 ml of physiological saline solution, 100 mg of the acid protease was 
dissolved. This solution was sterilized by filtering through a membrane 
filter. One ml of the filtrate was placed in a glass container sterilized 
in advance and then lyophilized. The container was then sealed to obtain 
lyophilized powder preparations. 
FORMULATION 2 
One hundred g of lyophilized acid protease, 97 g of lactose and 3 g of 
magnesium stearate were weighed and mixed to achieve homogeneity. Two 
hundred mg each of the resultant mixture was placed into No. 2 gelatin 
capsules and the capsules were coated with an enteric coating to give 
enteric capsules.