Polypeptide comprising repeated cell-adhesive core sequences

A polypeptide comprising repeated amino acid sequences of a cell-adhesive protein represented by the formula: EQU (Arg--Gly--Asp)n EQU or EQU (Tyr--Ile--Gly--Ser--Arg)n wherein n is a number ranging from 2 to 20; or a pharmaceutical acceptable salt thereof, which is valuable as an antimetastatic agent for cancer.

FIELD OF THE INVENTION 
This invention relates to a polypeptide comprising repetition of a certain 
amino acid sequence which consists of the adhesive core of cell-adhesive 
proteins, as well as to the use of said polypeptide in various drugs 
including an antimetastatic agent for cancer. 
PRIOR ART 
Some proteins such as fibronectin, laminin and vitronectin relate to the 
adhesion between cells and interstitial connective tissues and exert 
various physiological activities relating to the cellular functions of 
animal cells. These proteins are generally called cell-adhesive proteins. 
For example, fibronectin is a glycoprotein which is synthesized in liver 
and occurs at a concentration of approximately 0.3 mg/ml in human plasma. 
Fibronectin comprises a dimer of polypeptide chain A having a molecular 
weight of approximately 250 K and chain B having a molecular weight of 
approximately 240 K which are connected in the vicinity of each of the 
carboxyl-ends through disulfide bonds. Kornblihtt, A. R. et al. determined 
the primary structure of fibronectin by molecular cloning techniques [EMBO 
Journal, 4, 1755 (1985)]. On the other hand, the primary structure of 
laminin was determined by Sasaki, M. et al. [Proc. Natl. Acad. Sci. USA, 
84, 935 (1987); and J. Biol. Chem., 262, 17111 (1987)], and the primary 
structure of vitronectin was determined by Suzuki, S. et al. [EMBO 
Journal, 4, 2519 (1985)]. 
Further studies were made on the binding sites relating to cell-adhesion 
activity. The binding sites of both of the chains A and B were determined 
by partially digesting fibronectin with proteases and then examining the 
binding of the fragments thus obtained to heparin, collagen, cells and 
bacteria [Yamada, K. M., Ann. Rev. Biochem., 52, 761 (1983)]. Furthermore, 
it was clarified in 1984 that the core sequence of the cell-binding site 
of these chains was Arg-Gly-Asp (RGD) [Pierschbacher, M. D. et al., 
Nature, 309, 30 (1984)]. It has been known that this RGD sequence also 
occurs in other adhesive proteins such as vitronectin. It has been also 
clarified that the core sequence of the cell-binding site of laminin is 
Tyr-Ile-Gly-Ser-Arg [Graf, J. et al., Cell, 48, 989 (1987)]. 
Thus fibronectin and laminin adhere to receptors of cells via the 
above-mentioned core sequence and transmit some information to the cells 
to which they adhere. In addition, it is believed that these substances 
are capable of binding to biopolymers such as heparin, collagen and 
fibrin, and thus relate to the adhesion between cells and interstitial 
connective tissues and to the differentiation and growth of cells [Yamada, 
K. M., Ann, Rev. Biochem., 52, 761 (1983)]. 
Attempts have been made to apply these cell-adhesive proteins having the 
various biological activities described above to many purposes including 
medical ones. For example, a decrease in plasma fibronectin level would 
lower the reticuloendothelial function. This phenomenon is observed in, 
for example, septicemia induced by surgical operation or trauma, 
intravascular coagulation disseminata, burns, serious infectious diseases 
and surgical shock. It seems, therefore, that the administration of 
fibronectin is an effective way of ameliorating these conditions. Further 
fibronectin is expected to be applicable to the treatment of wounds and to 
immunomodulation, since it activates the migration of fibroblasts and 
macrophages. An attempt to apply fibronectin, which would promote the 
healing of wounds, to topical treatment of corneal disorders has already 
been made [Fujikawa, L. S. et al., Lab. Invest., 45, 120 (1981)]. 
On the other hand, laminin is capable of binding to collagen IV, heparin 
sulfate, proteoglycans and cells. Additionally, laminin exerts an effect 
of promoting the elongation of axons of neurons. Thus its effects in vivo 
are extremely interesting. 
Furthermore, cell-adhesive proteins have attracted attention since they 
relate to the metastasis of cancer. During the metastasis stage of cancer, 
cancer cells come in contact with various cells or biopolymers. When 
cell-adhesive proteins such as fibronectin or laminin are present at this 
stage, the cancer cells would form a multicellular mass, which allows 
growth or survival of the cancer cells. In fact, it has been observed that 
the administration of laminin mixed with cancer cells to an animal 
accelerated the metastasis of cancer. 
In contrast to these phenomena, however, it has been reported that a 
fragment obtained by digesting laminin with proteases acts to inhibit the 
metastasis of cancer [Barsky, S. H. et al., J. Clin, Invest., 74, 843 
(1984)]. It has likewise been confirmed that a tripeptide Arg-Gly-Asp 
corresponding to the adhesive core sequence of fibronectin [Humphries, M. 
J. et al., Science, 233, 467 (1986)] and a pentapeptide 
Tyr-Ile-Gly-Ser-Arg corresponding to that of laminin [Iwamoto, Y. et al., 
Science, 238, 1132 (1987)] act to inhibit the metastasis of cancer. 
As described above, cell-adhesive proteins such as fibronectin and laminin 
have various biological activities. Thus it is required to develop 
techniques for the application of substances related to them as drugs. It 
is believed that the antimetastatic effects of the adhesive core sequences 
of fibronectin and laminin are highly useful as drugs. However, the 
cell-adhesive activities of said core sequences are still unsatisfactory 
in practical applications from the medical viewpoint. Accordingly, it has 
been required to develop a substance exhibiting improved activity. 
However, these cell-adhesive proteins are natural substances and thus the 
supply thereof is restricted. In addition, it is considerably difficult to 
efficiently produce these glycoproteins through chemical synthesis or 
genetic engineering techniques. 
SUMMARY OF THE INVENTION 
The present invention provides a novel compound which sustains at an 
adequate level various biological activities observed in cell-adhesive 
proteins, which can be easily synthesized, and which causes no serious 
side effects on living organisms. The compound of the present invention is 
a polypeptide compound having high antimetastatic activity as compared 
with the above-mentioned core sequences. In addition to its antimetastatic 
activity, the compound of the present invention is also effective in 
immunomodulation, healing of wounds, inhibition of platelet agglutination 
and neuriatria. 
Accordingly, the present invention provides a novel polypeptide compound 
having a relatively low molecular weight which can be easily produced and 
has various activities similar to those of cell-adhesive proteins. 
Further, the present invention provides a novel polypeptide compound having 
a high antimetastatic activity. 
Furthermore, the present invention provides a pharmaceutical composition 
comprising said novel polypeptide compound.

DETAILED DESCRIPTION OF THE INVENTION 
The polypeptide of the present invention is characterized by comprising 
repeated adhesive core amino acid sequences of cell-adhesive proteins. In 
one embodiment, a preferable polypeptide compound of the present invention 
is represented by the following formula: 
EQU (Arg-Gly-Asp)n 
wherein n represents a number ranging from 2 to 20. Among the compounds of 
the above formula, those having a molecular weight of approximately 1,000 
to 10,000, in particular, approximately 1,500 to 5,000, are preferable, 
since they have sufficiently high biological activities and are highly 
soluble in aqueous solvents. 
In another embodiment, the compound of the present invention is represented 
by the following formula: 
EQU (Tyr-Ile-Gly-Ser-Arg)n 
wherein n represents a number ranging from 2 to 20. Among the compounds of 
the above formula, those having a molecular weight of approximately 5,000 
to 15,000, in particular, approximately 10,000, are preferable for the 
same reasons as those described above. 
Preparation 
The compound of the present invention may be prepared by polymerizing a 
corresponding short-chain peptide obtained in a conventional manner, for 
example, Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg by a continuous polymerization 
method utilizing diphenylphosphoryl azide (DPPA) [Nishi, N. et al., Int. 
J. Biol. Macromol., 2, 53 (1980); and Nishi N. et al., Int. J. Peptide 
Protein Res., 30, 275 (1987)]. 
Alternatively, the compound of the present invention may be obtained 
through genetic engineering techniques. 
The polypeptide compound of the present invention may consists of either L- 
or D-amino acids. Since the compound of the present invention is to be 
used as a drug, it may be converted into pharmaceutically acceptable 
salts, e.g., inorganic acid salts such as hydrochloride or sulfates or 
organic acid salts such as acetate, trifluoroacetate, lactate or tartrate. 
The conversion of the compounds into these salts may be conducted by 
utilizing conventional methods. 
ACTION 
The polypeptide compound of the present invention having the repeated 
cell-adhesive protein core sequences adheres to cells via said core 
sequences through the same mechanism as the one observed in the case of 
cell-adhesive proteins. Thus the compound of the present invention shows 
various biological activities as an agonist or an antagonist of said 
cell-adhesive proteins. It should be noted here that the compound of the 
present invention has an antimetastatic effect 6 to 10 times as high as 
that of the cell-adhesive protein core sequences. Furthermore, the 
polypeptide compound of the present invention has a wide range of 
biological activities. Namely, it is effective in immunomodulation, 
healing of wounds, inhibition of intravascular platelet aggregation caused 
by cancer cells and alleviation of neuro-disorders. It showed no toxicity 
when examined in experiments with mice. 
Accordingly, at least one of the polypeptide compounds of the present 
invention, optionally together with suitable conventional carriers or 
pharmaceutical adjuvants, can be administered to a patient as an 
antimetastatic agent for cancer, a remedy for wounds, an immunomodulator, 
a platelet aggregation inhibitor or an alleviator for neurodisorders. The 
dose may be determined depending on the condition, age, body weight etc. 
of the patient within the range of 0.2 .mu.g/kg to 400 mg/kg. 
The method of administering the polypeptide of the present invention may 
preferably be selected from those commonly used for peptide drugs, for 
example, parenteral administration methods such as intravenous, 
intramuscular or subcutaneous administration. The polypeptide compound of 
the present invention may be formulated into an injection suitable for the 
above-mentioned administration methods by, for example, the following 
method. It is dissolved in PBS or physiological saline solution, as will 
be shown hereafter, to thereby give an injection. Alternatively, it may be 
dissolved in, for example, a 0.1N aqueous solution of acetic acid and then 
lyophilized. These preparations may further contain conventional 
stabilizers such as glycine or albumin. Furthermore, collagen or a 
liposome may be used as a carrier in order to prolong the half-life of the 
compound of the present invention in blood. 
Alternatively, the polypeptide of the present invention may be formulated 
into microcapsules for oral administration by, for example, encapsulation 
in a liposome. Furthermore, it may be absorbed through mucosa other than 
the digestive tract by formulating into, for example, suppositories, 
sublingual tablets or nasal sprays. 
EVALUATION OF BIOLOGICAL ACTIVITY 
Now the biological activities of the compound of the present invention will 
be described based on the results of pharmacological tests. 
(1) Adhesiveness to Target Cells 
The adhesiveness to target cells of the compound of the present invention 
was examined in accordance with a method reported by Saiki et al. [Cancer 
Immunol., Immunother., 22, 125 (1986)]. Namely, a polypeptide of the 
present invention having a structure of (Arg-Gly-Asp)n and a molecular 
weight of approximately 5,000 prepared in the following Synthetic Example 
1 and a comparative polypeptide, (Arg, Gly, Asp)n, carrying a random 
sequence of the corresponding amino acids Arg, Gly and Asp and having a 
molecular weight of approximately 5,000 prepared in the following 
Synthetic Example 3 were employed. The adhesiveness of each compound was 
examined by using mouse Bl6-BL6 melanoma cells as the target. 
First, microculture wells were preliminarily coated with 20 .mu.g/ml of the 
polypeptide to be examined or 5 .mu.g/ml of mouse fibronectin (obtained 
from Seikagaku Kogyo, Japan) which was used as a positive control. Next 
0.05 ml/well of Bl6-BL6 melamona cells (2.times.10.sup.4) labeled with a 
radioactive iodide [.sup.125 I]IUdR was added thereto and the cells were 
cultured at 37.degree. C. for 20 minutes. The number of cells thus 
adsorbed by the culture wells was determined by measuring the 
radioactivity. 1% bovine serum albumin (BSA) was employed as a negative 
control. The Bl6-BL6 melanoma cells were labeled by culturing the cells to 
be labeled at the logarithmic growth phase for 24 hours in an MEM medium 
containing 5% of FBS and 0.3 .mu.Ci/ml of [.sup.125 I]IUdR (200 mCi/mmol; 
obtained from New England Nuclear, Boston, Mass., USA). 
The labeled cells were washed with physiological saline solution twice and 
suspended in 0.02% EDTA for 1 minute. Subsequently the cells were 
collected and suspended in a serum-free MEM medium. The cell suspension 
thus obtained was used in the above examination. The unadsorbed cells in 
the well were washed away with PBS 4 times. Then the cells adsorbed by the 
well were dissolved in 0.1 ml of 0.1N NaOH. The number of adsorbed cells 
was determined by measuring the radioactivity of the dissolved cells. 
Table 1 shows the results. As Table 1 indicates, the Poly(Arg-Gly-Asp) of 
the present invention and fibronectin accelerated the adsorption of the 
Bl6-BL6 melanoma cells, while the treatment with the Poly(Arg, Gly, Asp) 
having the random sequence and BSA caused scarcely any adsorption of the 
cells. These results indicate that the cell-adhesion activity of the 
polypeptide of the present invention is comparable to that of fibronectin. 
TABLE 1 
______________________________________ 
Adhesion of B16-BL6 melanoma cells to substrate 
coated with polypeptide or fibronectin 
Adhesiveness 
Incubated adhered cell No./ 
Coating with substrate (SD) 
______________________________________ 
Fibronectin 5849 .+-. 513 
Poly(Arg-Gly-Asp) 7967 .+-. 910 
Poly(Arg, Gly, Asp) 1750 .+-. 395 
BSA 1239 .+-. 347 
Fibronectin 
+ Arg-Gly-Asp 500 .mu.g/ml 
3708 .+-. 265 (37%) 
100 5320 .+-. 52 
+ His-Gly-Gly 500 6133 .+-. 787 
+ Poly(Arg-Gly-Asp) 
500 3715 .+-. 231 (36%) 
100 3687 .+-. 229 (37%) 
______________________________________ 
Table 1 also shows the results of examination of the cell adhesion 
specificities of the Poly(Arg-Gly-Asp) and fibronectin. Namely, 
2.times.10.sup.4 Bl6-BL6 cells were added to culture cells which had been 
treated in the same manner as the one described above. These cells were 
then cultured therein at 37.degree. C. for 20 minutes in the presence of 
100 or 500 .mu.g/ml of a synthetic peptide Arg-Gly-Asp, 100 or 500 
.mu.g/ml or the Poly(Arg-Gly-Asp) or 500 .mu.g/ml of His-Gly-Gly. It was 
thus found that the adhesion of the fibronectin to the cells was inhibited 
when they were cultured in the presence of the Arg-Gly-Asp or 
Poly(Arg-Gly-Asp) whereas no inhibition was observed in the presence of 
the His-Gly-Gly (refer to Table 1). 
The cell-adhesiveness of fibronectin was dose-dependently inhibited by 
using the synthetic tripeptide Arg-Gly-Asp. Furthermore, it was also 
inhibited by 5 mM EDTA. These results indicate that the cell adhesion 
mechanism of the Poly(Arg-Gly-Asp) would be specifically dependent on 
divalent metal ions such as calcium and magnesium ions and Arg-Gly-Asp 
sequence. Namely, it was suggested that the Poly(Arg-Gly-Asp) adheres to 
cells via the same receptor as the one which takes part in the adhesion of 
fibronectin to cells. 
Thus the results of the above examination suggested that the polypeptide 
compound of the present invention may be applicable to drugs as an agonist 
or an antagonist of fibronectin. 
(2) Antimetastatic Effect 
(a) Next, the antimetastatic effect on cancer of the compound of the 
present invention having repeated core sequences Arg-Gly-Asp was examined. 
A 500 .mu.g portion each of a synthetic polypeptide Poly(Arg-Gly-Asp) 
having a molecular weight of approximately 5,000, a tripeptide Arg-Gly-Asp 
and a polypeptide Poly(Arg, Gly, Asp) having a molecular weight of 
approximately 5,000 was mixed with 5.times.10.sup.4 Bl6-BL6 melanoma cells 
which were in the above-mentioned logarithmic growth phase and showed 
extremely strong metastasis, in PBS. 0.2 ml of each mixture thus obtained 
was intravenously injected into a group of male C57BL/6 mice. Each group 
consisted of five animals. Fourteen days after the administration, the 
cancer colonies in the lungs of the animals were counted and the obtained 
data were compared with those of a control group which received PBS alone. 
The results are given in Table 2-1 (Test 1). As the results indicate, the 
metastasis of cancer to lungs was remarkably inhibited by the 
administration of the Poly(Arg-Gly-Asp). In contrast thereto, the 
administration of either Arg-Gly-Asp or Poly(Arg, Gly, Asp) showed no such 
antimetastatic effect. Table 2-1 further indicates that the amount of 
Arg-Gly-Asp needed to achieve an antimetastatic effect comparable to that 
of the Poly(Arg-Gly-Asp) is 3,000 .mu.g, i.e., approximately 6 times as 
much as that of the polypeptide compound of the present invention. 
(b) Next, the antimetastatic activities of two polypeptides each having 
repeated Arg-Gly-Asp sequences, namely, Poly(Arg-Gly-Asp)-1500 
(approximate molecular weight: 1,500) and Poly(Arg-Gly-Asp)-5000 
(approximate molecular weight: 5,000) were examined. 1,000 .mu.g of each 
compound was administered to mice in the same manner as described in the 
above Test 1 and the effects were observed. As Table 2-1 (Test 2) 
indicates, both of the compounds used in Test 2 showed a remarkable 
antimetastatic effect compared with the control group. 
Furthermore, Poly(Arg-Gly-Asp)-5000 was not mixed with Bl6-BL6 cells but 
intravenously administered to mice to which Bl6-BL6 cells had been 
administered 5 minutes before. In this case also, a high antimetastatic 
effect was observed. This fact indicates that the administration of the 
compound of the present invention in an appropriate manner, such as 
intravenous injection, would give an antimetastatic effect. 
TABLE 2-1 
______________________________________ 
Effect of polypeptides on experimental metastasis of 
cancer induced by injecting B16-BL6 melanoma cells 
Adminis- Dose Metastasis to lung 
Compound tration (.mu.g) 
mean .+-. SD (range) 
______________________________________ 
Test 1 
PBS (untreated) -- 91 .+-. 19 (64-112) 
Poly(Arg-Gly-Asp) 
simultaneous 
500 23 .+-. 3 (20-28)* 
Arg-Gly-Asp " 3000 18 .+-. 24 (1-52)* 
500 46 .+-. 14 (33-68) 
Poly(Arg, Gly, Asp) 
" 500 65 .+-. 4 (64-68) 
Test 2 
PBS (untreated) -- 52 .+-. 8 (40-60) 
Poly(Arg-Gly-Asp) 
simultaneous 
1000 6 .+-. 4 (2-14)* 
(m.w.: 5,000) 
separate 1000 16 .+-. 4 (10-20)* 
Poly(Arg-Gly-Asp) 
simultaneous 
1000 14 .+-. 4 (10-18)* 
(m.w.: 1,500) 
______________________________________ 
*p &lt; 0.001 when compared with untreated control in Student's tcalibration 
 
(c) Next, the antimetastatic effect of a polypeptide compound 
Poly(Tyr-Ile-Gly-Ser-Arg) obtained in Synthetic Example 4 described 
hereafter, which had a molecular weight of approximately 10,000 and a 
cell-adhesive core sequence of laminin (Tyr-Ile-Gly-Ser-Arg), was also 
examined. Namely, portions of 2, 20 and 100 .mu.g of the above compound 
were each mixed with 3.times.10.sup.4 Lewis lung cancer cells (3LL) and 
administered to a group of C57BL/6 mice in the same manner as the one 
described in the above (b). Thus the antimetastatic effect of this 
compound was examined. Table 2--2 shows the results. As Table 2-2 
indicates, the metastasis of cancer to the lungs was remarkably inhibited 
by the administration of the Poly(Tyr-Ile-Gly-Ser-Arg). In contrast 
thereto, the administration of 100 .mu.g of a pentapeptide 
Tyr-Ile-Gly-Ser-Arg showed scarcely any antimetastatic effect. Though this 
result is not shown in the above Table, it was required to administer at 
least 200 .mu.g of this compound in order to achieve a significant effect. 
These results indicate that the polypeptide of the present invention 
having the repeated pentapeptide sequences has an antimetastatic activity 
almost 10 times as high as that of the original pentapeptide. 
TABLE 2-2 
______________________________________ 
Effect of polypeptides on experimental metastasis 
induced by injecting metastatic cancer cells 
Dose No. of instances of 
Administered (.mu.g/ metastasis to lung 
Cancer 
compound animal) mean .+-. SD (range) 
______________________________________ 
B16- Untreated (PBS) -- 115 .+-. 24 (86-149) 
BL6 Poly(Tyr-Ile-Gly-Ser-Arg) 
100 19 .+-. 9 (10-32)* 
20 43 .+-. 13 (24-56)* 
5 81 .+-. 9 (67-89)** 
Tyr-Ile-Gly-Ser-Arg 
100 101 .+-. 17 (77-122) 
20 109 .+-. 39 (61-164) 
5 128 .+-. 31 (104-180) 
3LL Untreated (PBS) -- 139 .+-. 19 (113-158) 
Poly(Arg-Gly-Asp) 
500 34 .+-. 7 (26-41)* 
Poly(Tyr-Ile-Gly-Ser-Arg) 
100 14 .+-. 11 (2-28)* 
Tyr-Ile-Gly-Ser-Arg 
100 123 .+-. 45 (74-187) 
______________________________________ 
*p &lt; 0.001 when compared with untreated control in Student's tcalibration 
**p &lt; 0.02 when compared with untreated control in Student's tcalibration 
 
(3) Inhibitory Effect on Cancer Cell Retention in Lungs 
An examination similar to the one described above proved that the compound 
of the present invention has an inhibitory effect on the retention of 
cancer cells in lungs. Bl6-BL6 cells at the logarithmic growth phase were 
labeled with [.sup.125 I]IUdR. 0.2 ml portions of a solution containing 
2.times.10.sup.4 /mouse of these labeled cells and 500 .mu.g/mouse of 
Poly(Arg-Gly-Asp) having a molecular weight of 5,000 were intravenously 
injected in the tails of C57BL/6 mice. The liver, kidneys, spleen and 
lungs of each animal were removed 30 minutes or 24 hours after the 
administration and the radioactivity in these organs was determined. Mice 
which received PBS were used as control animals. Table 3 shows the 
results. The radioactivity of the lungs of the Poly(Arg-Gly-Asp) 
administration group was markedly lower than that of the control group, 
though no significant difference was observed in other organs including 
blood. This fact indicated that the retention of the cancer cells in the 
lungs was significantly inhibited in the test group. 
TABLE 3 
______________________________________ 
Distribution and retention of [.sup.125 I]IUdR-labeled 
B16-BL6 melanoma cells administered together with 
Poly(Arg-Gly-Asp) to C57Bl/6 mice 
Radioactivity (cpm .+-. SD)* 
Organ Untreated (PBS) Poly(Arg-Gly-Asp) 
______________________________________ 
Lung 2034 .+-. 284 
(13.2%)** 1121 .+-. 281 
(7.3%)*** 
Liver 260 .+-. 30 
(1.7%) 195 .+-. 42 
(1.3%) 
Spleen 38 .+-. 12 
(0.2%) 59 .+-. 7 
(0.4%) 
Kidney 78 .+-. 2 
(0.5%) 73 .+-. 19 
(0.5%) 
Blood (1 ml) 
560 .+-. 260 
(3.7%) 304 .+-. 94 
(2.0%) 
______________________________________ 
*Average of 3 animals. 
**Percentage relative to the administered radioactivity (15308 .+-. 1605 
cpm/2 .times. 10.sup.4 cells) is given in parenthesis. 
***p &lt; 0.02 in Student's tcalibration when compared with the untreated 
(PBS) group. 
(4) Antimetastatic Effect on Spontaneous Metastasis Model 
Furthermore, the antimetastatic effect of the compound of the present 
invention on a spontaneous metastasis model was examined. Namely, Bl6-BL6 
cells were transplanted into the foot pads of C57BL/mice (each group 
having five animals). Then 100 .mu.g or 50 .mu.g of Poly(Arg-Gly-Asp) 
having a molecular weight of approximately 5,000 was topically 
administered directly to the site of the transplanted cancer in each 
animal in a single or divided dose within a determined period. The 
transplanted cancer was removed on the 21st day and the mouse was 
anatomized 2 weeks later to examine the metastasis of the cancer into the 
lungs. Table 4 shows the results. It was found that the metastasis of the 
cancer to the lungs was remarkably reduced, though the growth of the 
transplanted cancer per se could not be suppressed, by administering 100 
.mu.g of the compound on the 1st or 7th day in a single dose or by 
administering 50 .mu.g portions of the same on the 7th, 10th, 13th and 
16th days. 
In contrast, the administration of 100 .mu.g of Poly(Arg, Gly, Asp) having 
a random amino acid sequence on the 7th day did not suppress the 
metastasis of cancer into the lungs at all. 
TABLE 4 
______________________________________ 
Inhibitory effect of polypeptides on spontaneous metastasis 
of B16-BL6 melanoma cells administered to foot pad 
Observation on 21st day 
Size of Number of 
trans- metastatic 
Dose Adminis- planted cancer 
(.mu.g/ tration cancer to lung 
Compound animal) date (mm .+-. SD) 
(mm .+-. SD) 
______________________________________ 
Untreated 10 .+-. 4 
129 .+-. 38 
(PBS) (78-180) 
Poly- 100 1 8 .+-. 4 24 .+-. 17 
(Arg-Gly-Asp) (4-49)* 
7 9 .+-. 3 29 .+-. 22 
(0-50)* 
14 10 .+-. 4 
120 .+-. 29 
(79-155) 
20 8 .+-. 3 131 .+-. 79 
(75-247) 
50 .times. 4 
7, 10, 8 .+-. 3 26 .+-. 29 
13, 16 (0-68)* 
Poly 100 7 11 .+-. 3 
158 .+-. 62 
(Arg, Gly, Asp) (117-230) 
______________________________________ 
*p &lt; 0.001 in Student's tcalibration when compared with untreated control 
 
(5) Suppressive Effect on Platelet Aggregation Indicated by Cancer Cells 
The effect of Poly(Arg-Gly-Asp) on the platelet aggregation induced by 
cancer cells was examined. The blood of a C57BL/6 mouse was centrifuged at 
160.times.g for 15 minutes to give platelet rich plasma (PRP). As a 
control, platelet poor plasma (PPP) obtained by centrifuging the blood at 
1,000.times.g for 10 minutes was employed. Poly(Arg-Gly-Asp) having a 
molecular weight of 5,000 was added to 250 .mu.l of the above PRP or PPP 
plasma (5.times.10.sup.5 /.mu.l). After preincubation for 5 to 7 minutes, 
the mixture was added to a suspension of Bl6-BL6 cells in physiological 
saline (approximately 10.sup.6 /ml) and stirred at 37.degree. C. at 1,000 
rpm. During this period, the aggregation of the platelets was monitored 
with a dual aggregometer (Model 440; Chrono-Log, USA). For comparison, the 
above procedure was repeated replacing the Poly(Arg-Gly-Asp) with 
Poly(Arg, Gly, Asp). FIGS. 1 to 3 together show the results. FIGS. 1, 2 
and 3 respectively show the platelet aggregation observed when the 
heparinized PRP was treated with PBS; with 100 .mu.g/ml of Poly(Arg, Gly, 
Asp); and with 100 .mu.g/ml of Poly(Arg-Gly-Asp); 7 minutes prior to the 
addition of the Bl6-BL6 melanoma cells (shown by an arrow) in each case. 
As these figures indicate, the compound of the present invention 
Poly(Arg-Gly-Asp) completely suppressed the platelet aggregation (FIG. 3), 
while the comparative one Poly(Arg, Gly, Asp) showed scarcely any 
suppression capability (FIG. 2). 
(6) Toxicity 
The above tests (1) to (5) provided that the Poly(Arg-Gly-Asp) having a 
molecular weight of 5,000 or 1,500 and the Poly(Tyr-Ile-Gly-Ser-Arg) 
having a molecular weight of 10,000 showed neither any cytotoxicity on 
erythrocytes, spleen and thymus cells nor any undesirable aggregation 
effect on serum proteins. 
FORMULATION 
As stated above, the polypeptide of the present invention is, like other 
pharmaceutical peptides, preferably administered parenterally in the form 
of an injection for intravenous, intramascular or subcutaneous 
administration. Alternatively, the peptide can be administered orally in 
the form of a microcapsule containing the peptide in liposome or can be 
administered by absorption through mucous membranes other than digestive 
tracts in the form of a suppository, sublingual agent or nasal spray. 
Following formulations are given as non-limiting examples of the invention. 
A) Injectable solution 
______________________________________ 
Composition 
______________________________________ 
Peptide of the invention 
2 g 
Sodium chloride 8 g 
Ascorbic acid 2 g 
Sterile water 1 l 
______________________________________ 
The peptide, ascorbic acid and sodium chloride were dissolved in sterile 
water, an ampule was filled with 5 ml of the solution and then sealed to 
afford an injectable solution. 
B) Lyophilizate 
______________________________________ 
Composition 
______________________________________ 
Peptide of the invention 
2 g 
Sorbitol 20 g 
______________________________________ 
The peptide and sorbitol were dissolved in 200 ml of sterile water, a vial 
was filled with 1 ml of the solution and lyophilized, and the vial was 
then sealed. This composition can be dissolved in 5 ml of sterile water 
before parenteral administration. 
Synthetic Example 
Now examples of the process of preparing the compound of the present 
invention will be described. Amino acid derivatives were purchased from 
Peptide Institute, Inc. (Hinoh-shi, Osaka, 562 Japan), while peptides were 
synthesized by the liquid phase method. The evaluation of the purity of 
polypeptides and the identification of each compound were conducted by 
thin layer chromatography [developing solvent: (A) the upper layer of a 
mixture of n-butanol:acetic acid:water=4:1:5; (B) 
n-butanol:pyridine:acetic acid:water=15:10:3:12; (C) aqueous 
ammonium-saturated n-butanol etc.], elementary analysis and infrared 
spectrometry. Polypeptides having regular sequences or random sequences 
were synthesized by the method based on use of diphenylphosphoryl azide 
(DPPA). The protecting groups (Mts and Bzl groups) of the side-chain 
functional groups were removed by utilizing methanesulfonic acid/anisole 
or trifluoromethanesulfonic acid/thioanisole. Finally, the guanide group 
in the arginine side chain was converted into hydrochloride with an ion 
exchange resin (Amberlite IRA-400). The removal of the protecting groups 
was confirmed by infrared spectrometry. The molecular weight was roughly 
estimated by polyacrylamide gel electrophoresis (gel concentration: 15% or 
20%) in the presence of 1% of sodium dodecyl sulfate. 
Synthetic Example 1 
Polypeptide (m.w.: ca. 5,000) 
(1) t-Butoxycarbonylglycyl-.beta.-benzyl-L-aspartic acid 
[Boc-Gly-Asp(OBzl)-OH] (I) 
5.3 g (30 mmol) of t-butoxycarbonyl glycine was dissolved in 100 ml of 
purified THF and 4.6 ml (33 mmol) of triethylamine and 4.33 ml (33 mmol) 
of isobutyl chloroformate) were added thereto at -15.degree. C. The 
mixture was stirred at this temperature for 10 minutes. To the resultant 
solution of a mixed acid anhydride was added a solution which had been 
prepared by dissolving 8.0 g (36 mmol) of .beta.-benzyl-L-aspartic acid 
and 5.02 ml (36 mmol) of triethylamine in water and cooling to 0.degree. 
C. The mixture was then stirred at 0.degree. C. for 1 hour and further at 
room temperature for 15 hours. After removing the THF by concentration 
under reduced pressure, cold 10% citric acid was added thereto. The 
precipitate thus formed was extracted with ethyl acetate twice. The 
extracts were combined and washed 5 times with one fifth the volume of 5% 
citric acid and then 10 times with water (one tenth the volume thereof). 
The solution was dehydrated over anhydrous sodium sulfate and concentrated 
to dryness. Then the residue was dissolved in ether and precipitated with 
n-hexane. After repeating this precipitation procedure 3 times, the 
obtained product was dried, whereby 6.9 g of the titled compound was 
obtained. 
(2) Glycyl-.beta.-benzyl-L-aspartate hydrochloride [HCl.H-Gly-Asp(OBzl)-OH] 
(II) 
3.2 g of compound (I) as obtained in the above step (1) was dissolved in 80 
ml of purified dioxane. After adding 80 ml of 4N HCl/dioxane, the mixture 
was stirred at room temperature for 1 hour. After concentrating to 
dryness, dry ether was added thereto whereby the product was crystallized, 
which was then collected by centrifugation and washed with dry ether 
several times. Thus 2.4 g of the titled compound was obtained. 
(3) N.sup..alpha. -t-butoxycarbonyl-N.sup..omega. 
-mesitylenesulfonyl-L-arginylglycyl-.beta.-benzyl-L-aspartate 
[Boc-Arg(Mts)-Gly-Asp(OBzl)-OH] (III) 
3.4 g (7.4 mmol) of N.sup..alpha. -t-butoxycarbonyl-N.sup..omega. 
-mesitylenesulfonyl-L-arginine was dissolved in 70 ml of purified THF and 
1.13 ml (8.1 mmol) of triethylamine and 1.07 ml (8.1 mmol) of isobutyl 
chloroformate were added thereto at -15.degree. C. The resultant mixture 
was stirred at this temperature for 10 minutes to allow a mixed acid 
anhydride to be formed. 2.4 g (approximately 8.0 mmol) of compound (II) as 
described in the above step (2) and 2.68 ml (19.2 mmol) of triethylamine 
were dissolved in a solvent mixture comprising 70 ml of THF, 50 ml of DMF 
and 10 ml of water and cooled to 5.degree. C. This solution was added to 
the above-mentioned mixed acid anhydride solution and the mixture was 
stirred at 5.degree. C. for 1 hour followed by stirring at room 
temperature for 15 hours. After removing the THF and water by 
concentration under reduced pressure, 5% citric acid was added to the 
residual DMF solution. The precipitate thus formed was separated by 
decantation, thoroughly washed with 5% citric acid and water and dried. 
After washing with ether twice, the residue was reprecipitated from 
methanol/ether and separated by decantation. The precipitate was converted 
into a powder by treating with ether. This powder was then centrifuged, 
washed with ether and dried. Thus 3.9 g of the titled compound was 
obtained. 
(4) N.sup..omega. 
-mesitylenesulfonyl-L-arginylglycyl-.beta.-benzyl-L-aspartate 
hydrochloride [HCl.H-Arg(Mts)-Gly-Asp(OBzl)-OH] (IV) 
1.5 g (2.1 mmol) of compound (III) was dissolved in 30 ml of purified 
dioxane and 30 ml of 4N HCl/dioxane was added thereto. The mixture was 
stirred for 1 hour. After concentrating to dryness, the product was 
crystallized by adding dry ether. The crystals were centrifuged, washed 
with dry ether several times and dried. Thus 1.3 g of the titled compound 
was obtained. 
(5) L-arginylglycylaspartate hydrochloride (2HCl.H-Arg-Gly-Asp-OH) (V) 
300 mg of compound (III) was dissolved in a mixture of 4 ml of 
methanesulfonic acid and 1 ml of anisole and the resultant solution was 
stirred at room temperature for 1.5 hours. After adding dry ether, the 
precipitate thus formed was separated by decantation, thoroughly washed 
with dry ether and dried. The product was dissolved in a small amount of 
water and passed through a column packed with Amberlite IRA-400 (Cl form). 
Then a fraction containing the target compound was concentrated to dryness 
and the residue was crystallized by treating with methanol/ether. After 
centrifuging, washing with ether and drying, 117 mg of the titled compound 
was obtained. 
(6) Poly(N.sup..omega. 
-mesitylenesulfonyl-L-asparginylglycyl-.beta.-benzyl-L-aspartate) 
[Poly(Arg(Mts)-Gly-Asp(OBzl)] (VI) 
400 mg (0.58 mmol) of the compound (IV) was dissolved in 1.2 ml of purified 
DMSO and 0.19 ml (0.87 mmol) of DPPA and 0.285 ml (2.03 mmol) of 
triethylamine were added thereto. The obtained mixture was stirred at 
5.degree. C. to 8.degree. C. for 1 hour followed by stirring at room 
temperature for 2 days. Next, DPPA and triethylamine were again added 
thereto each in the same amount. The polymerization was continued for a 
further 2 days. The polymer thus formed was precipitated with water, 
centrifuged, thoroughly washed with water and methanol, and dried. Thus 
295 mg of the titled compound was obtained. 
(7) Poly(L-arginylglycyl-L-aspartate hydrochloride) [Poly(Arg-Gly-Asp)HCl] 
(VII) 
100 mg of compound (VI) was treated with a mixture of 2 ml of 
methanesulfonic acid and 0.4 ml of anisole in order to remove the 
protecting group in the side chain. Then the position was converted into 
the hydrochloride by treating with an anion exchange resin. The process 
used for the synthesis of compound (V) was repeated. Thus 60 mg of the 
titled compound having a molecular weight of approximately 5,000 was 
obtained. 
SYNTHETIC EXAMPLE 2 
Comparative Compound with Random Sequence 
(1) Copoly(N.sup..omega. -mesitylenesulfonyl-L-arginine, glycine, 
.beta.-benzyl-L-asparate) [Copoly(Arg(Mts), Gly, Asp(OBzl)] (VIII) 
A mixture comprising 356 mg (1.0 mmol) of N.sup..omega. 
-mesitylenesulfonyl-L-arginine, 75 mg (1.0 mmol) of glycine and 223 mg 
(1.0 mmol) of .beta.-benzyl-L-aspartate was polymerized with 0.97 ml (4.5 
mmol) of DPPA and 1.05 ml (7.5 mmol) of triethylamine in 2.0 ml of 
purified DMSO. The process employed for the synthesis of compound (VI) was 
repeated. 
(2) Copoly(L-arginine hydrochloride, glycine, L-aspartic acid) [Copoly(Arg, 
Gly, Asp)HCl] (IX) 
100 mg of compound (VIII) was treated with a mixture of 2 ml of 
methanesulfonic acid and 0.4 ml of anisole to remove the protecting groups 
in the side chain. Then the positions were converted into the 
hydrochlorides by treating with an ion exchange resin. The process 
employed for the synthesis of compound (V) was repeated. Thus 50 mg of the 
titled compound having a molecular weight of approximately 5,000 was 
obtained. 
SYNTHETIC EXAMPLE 3 
Polypeptide (m.w.: ca. 1,500) 
Poly(L-arginyl-glycyl-L-aspartic acid)hydrochloride 
[Oligo(Arg-Gly-Asp).HCl] (X) 
The polymerization of compound (VI) was ceased 90 minutes after initiation. 
The oligopeptide having a protected side chain thus obtained was treated 
in the same manner as the one described in the synthesis of compound 
(VII). Thus the titled compound having an average molecular weight of 
approximately 1,500 was obtained. 
SYNTHETIC EXAMPLE 4 
Polypeptide (m.w.: ca. 10,000) 
(1) N.sup..omega. -mesitylenesulfonyl-L-arginine-O-methylester 
hydrochloride [HCl-Arg(Mts)-OMe] (XI) 
2.10 g of N.sup..omega. -t-butoxycarbonyl-N.sup..omega. 
-mesitylenesulfonyl-L-arginine [Boc-Arg(Mts)-OH] was dissolved in 31 ml of 
dry dioxane and 31 ml of 4N HCl-dioxane was added thereto. The obtained 
mixture was stirred at room temperature for 1 hour. The resulting solution 
was concentrated under reduced pressure to dryness and then crystallized 
by adding dry ether. The target product was collected by centrifugation, 
washed with dry ether several times and dried in a desiccator. Thus 1.1 g 
of HCl.H-Arg(Mts)-OH from which Boc groups had been removed was obtained. 
This product was next added to a solution, which had been prepared by 
slowly adding 2.9 ml of SOCl.sub.2 to 10.6 ml of methanol and stirring at 
-10.degree. C. for 10 minutes, and the obtained mixture was stirred for 15 
hours, concentrated to dryness, crystallized from dry ether, centrifuged, 
washed with dry ether several times and dried. Thus 0.94 g of 
HCl.H-Arg(Mts)-OMe was obtained. 
(2) N.sup..omega. -t-butoxycarbonyl-.beta.-benzyl-L-seryl-N.sup..omega. 
-mesitylenesulfonyl-L-arginine methyl ester [Boc-Ser(Bzl)-Arg(Mts)-OMe] 
(XII) 
0.69 g (2.3 mmol) of Boc-Ser(Bzl)-OH and 0.94 g (2.3 mmol) of 
HCl-Arg(Mts)-OMe were dissolved in a mixture of 16.3 ml of purified DMF 
and 16.3 ml of dioxane and the obtained solution was stirred at 0.degree. 
C. for 10 minutes. Then 0.76 ml (2.76 mmol) of DPPA and 0.74 ml (7.36 
mmol) of TEA were added thereto and the mixture was stirred at room 
temperature for 24 hours. This solution was concentrated under reduced 
pressure and the dioxane was distilled off. An aqueous solution of NaCl 
was added to the residue and the precipitate thus formed was separated by 
decantation and washed with water. The residue was extracted with ethyl 
acetate and the extract was thoroughly washed with 5% citric acid, water 
and sodium bicarbonate, dehydrated over anhydrous sodium sulfate (Na.sub.2 
SO.sub.4), and concentrated to dryness. 
(3) Boc.Gly-Ser(Bzl)-Arg(Mts)-OMe (XIII) 
1.03 g of the compound (XII) was dissolved in 25.3 ml of dry dioxane and 
25.3 ml of 4N HCl/dioxane was added thereto. The obtained solution was 
stirred at room temperature for 1 hour and then concentrated to dryness. 
It was then crystallized by adding dry ether and the crystals were 
centrifuged, washed with dry ether several times and dried. Thus 0.68 g 
(1.2 mmol) of HCl.H-Ser(Bzl)-Arg(Mts)-OMe was obtained. This compound was 
dissolved in a mixed solvent of 9.4 ml of purified DMF and 9.4 ml of 
dioxane, together with 0.26 g (1.5 mmol) of Boc-Gly-OH. After stirring at 
0.degree. C. for 10 minutes, 0.5 ml (1.8 mmol) of DPPA and 0.49 ml (4.8 
mmol) of triethylamine were added thereto and the obtained mixture was 
stirred at room temperature for 24 hours. The solution thus obtained was 
subsequently treated in the same manner as in the synthesis of compound 
(XII), i.e. the solution was concentrated under reduced pressure and an 
aqueous solution of NaCl was added thereto. The precipitate thus formed 
was washed and dried in order that acidic and alkaline materials would be 
removed. Thus 0.48 g of Boc-Gly-Ser(Bzl)-Arg(Mts)-OMe was obtained. 
(4) Boc-Ile-Gly-Ser(Bzl)-Arg(Mts)-OMe (XIV) 
The Boc group of product (XIII) was removed by the same method as described 
in regard to compound (XI) to give 0.40 g (0.63 mmol) of 
HCl.H-Gly-Ser(Bzl)-Arg(Mts)-OMe. It was then dissolved in a mixed solvent 
of 5.4 ml of rectified DMF and 5.4 ml of dioxane together with the 
equimolar amount (0.14 g) of Boc-Ile-OH. The resulting solution was 
stirred at 0.degree. C. for 10 minutes and then 0.21 ml of DPPA and 0.20 
ml of triethylamine were added. The mixture was stirred at room 
temperature for 24 hours. The solution thus obtained was freed from any 
acidic and alkaline materials in the same manner as described in regard to 
compounds (XII) and (XIII). After drying, 0.28 g of 
Boc-Ile-Gly-Ser(Bzl)-Arg(Mts)-OMe was obtained. 
(5) Boc-Tyr(Bzl)-Ile-Gly-Ser(Bzl)-Arg(Mts)-OMe (XV) 
The Boc group of the product (XIV) was removed in the same manner as 
described in (1), except that approximately 2 or 3 times as much solvent 
was employed and the reaction time was extended to approximately 2 to 3 
times in consideration of the length of the peptide chain and the steric 
hindrance of the Ile side chain. Namely, 0.28 g of the product (XIV), 6.0 
ml of dry dioxane and 6.0 ml of 4N HCl/dioxane were employed while 
stirring was being conducted for a period of 2 hours. The resultant 
peptide [HCl.H-Ile-Gly-Ser(Bzl)-Arg(Mts)-OMe, yield: 0.14 g (0.19 mmol)] 
was dissolved in a mixture of 2.8 ml of rectified DMF and 2.8 ml of 
dioxane, together with 0.14 g (0.19 mmol) of Boc-Tyr(Bzl)-OH. The solution 
was stirred at 0.degree. C. for 10 minutes. Next, 0.12 ml of DPPA and 0.72 
ml of triethylamine were added thereto and the mixture was stirred at room 
temperature for 24 hours. The solution thus obtained was washed and 
dried, in the same manner as described regarding compound (XII). Thus 0.12 
g of Boc-Tyr(Bzl)-Ile-Gly-Ser(Bzl)-Arg(Mts)-OMe was obtained. 
(6) 0.14 g (0.13 mmol) of the product (XV) was dissolved in 1.95 ml of dry 
dioxane and 0.65 ml of 1N NaOH was added thereto. The mixture was stirred 
at room temperature for 3 hours. After removing the dioxane by 
concentrating under reduced pressure, approximately 20 ml of water was 
added and a small amount of insoluble matter was filtered off. The 
filtrate was cooled and 10% citric acid at 0.degree. C. was added thereto. 
The precipitate thus formed was filtered, washed with a small amount of 
water twice and then dried. Thus 0.10 g of 
Boc-Tyr(Bzl)-Ile-Gly-Ser(Bzl)-Arg(Mts)-OH was obtained. This product was 
stirred in 6.0 ml of dry dioxane and 6.0 ml of 4N HCl/dioxane for 3 hours 
to remove the Boc group. Thus 0.08 g of 
HCl.H-Tyr(Bzl)-Ile-Gly-Ser(Bzl)-Arg(Mts)-OH was obtained. 
(7) Poly(Try-Ile-Gly-Ser-Arg)HCl (XVII) 
60 mg (0.061 mmol) of HCl.H-Tyr(Bzl)-Ile-Gly-Ser(Bzl)-Arg(Mts)-OH was 
dissolved in 0.2 ml of rectified DMSO and 0.07 ml of DPPA and 0.06 ml of 
triethylamine were added thereto. The solution was stirred at 0.degree. C. 
for 1 hour followed by stirring at room temperature for 48 hours. After 48 
hours, additional amount of DPPA and triethylamine, each in the same 
quantity as specified above, were added thereto and the mixture was 
stirred at 0.degree. C. for 1 hour followed by stirring at room 
temperature for 48 hours. Water was added to the solution thus obtained 
and the mixture was centrifuged several times to remove the DPPA etc. 
After adding a small amount of methanol, centrifuge was repeated four more 
times to remove low molecular weight peptides dissolved in the methanol. 
Then the residue was centrifuged again by being dispersed in ether and 
dried. In order to remove the protecting groups in the side chains, a 40 
mg portion of the resulting Poly(Tyr(Bzl)-Ile-Gly-Ser(Bzl)-Arg(Mts)) was 
added to a solvent mixture comprising 2 ml of trifluoroacetic acid and 0.4 
ml of trifluoromethanesulfonic acid, together with 1 ml of thioanisole 
which was used as a scavenger. The mixture was stirred at 0.degree. C. 
for 2 hours. The resulting solution was concentrated to dryness and dry 
ether was added thereto. The precipitate thus formed was wasahed with 
ether 4 times and dried. It was next dissolved in a small amount of water 
and passed through a column of an anion exchange resin (Amberlite IRA-400, 
Cl-form). The fraction containing the target product was concentrated to 
dryness and the residue was precipitated with a small amount of methanol. 
After washing with ether several times and drying, 22 mg of 
Poly(Tyr-Ile-Gly-Ser-Arg)HCl was obtained. This polypeptide had a 
molecular weight of approximately 10,000. 
As described above, the novel polypeptide compound of the present invention 
having the repeated structure has a higher cell-adhesiveness, various 
biological activities including an antimetastatic effect, and scarcely any 
toxicity, as compared with the core sequences of cell-adhesive proteins. 
Furthermore, the polypeptide compound of the present invention has a 
relatively simple structure, which makes the synthesis thereof easy. Thus 
it is highly valuable as a drug.