M-CSF as a therapeutic agent for thrombocytopenia

A therapeutic agent for thrombocytopenia due to hemopoietic disorder induced by various causes comprises as an active ingredient a specific human monocytemacrophage colony stimulating factor. It is administered for the treatment and/or prevention of such thrombocytopenia.

FIELD OF THE INVENTION 
This invention relates to a therapeutic agent for thrombocytopenia due to 
hemopoietic disorder induced by various causes. More particularly, the 
invention relates to a therapeutic agent for thrombocytopenia which 
comprises a human monocyte-macrophage colony stimulating factor, which is 
one of human hemopoietic factors, as an active ingredient. 
BACKGROUND OF THE INVENTION 
Platelet transfusion is a powerful means for treating patients either 
actually suffering from or facing a high risk of severe hemorrhage due to 
marked decrease in the number of platelets or decreased hemopoietic 
function caused by various types of hemopoietic disorder. However, from 
the medical practice viewpoint, the current situation is not such that 
platelet preparations are available promptly in sufficient quantities. 
Moreover, the risk of patients being infected with such pathogenic viruses 
as ATL (adult T cell leukemia) or AIDS (acquired immune deficiency 
syndrome) on the occasion of platelet transfusion is remarkably high. 
SUMMARY OF THE INVENTION 
As a result of investigations made in an attempt to find out a means of 
promoting platelet formation in patients suffering from or facing a high 
risk of severe hemorrhage in the course of chemotherapeutic or 
radiotherapeutic treatment of leukemia or malignant tumor, or suffering 
from aplastic anemia, the present inventors found that administration of a 
preparation comprising a specific human monocyte-macrophage colony 
stimulating factor (CSF) as an active ingredient in the course of 
chemotherapy can result in rapid restoration of a normal platelet level 
and have now completed the present invention based on this finding. The 
instant invention provides a therapeutic agent for thrombocytopenia which 
is to be administered for the treatment and/or prevention of hemopoietic 
disorder-induced thrombocytopenia, said agent comprising as an active 
ingredient a human monocytemacrophage colony stimulating factor wherein 
said human monocytemacrophage colony stimulating factor has the following 
physicochemical properties a) to f): 
a) Molecular weight 
It is a homodimer composed of two identical subunits and, when determined 
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 
the molecular weight of each subunit resulting from dissociation with a 
reducing agent is 35,000-45,000 daltons, 
b) Isoelectric point 
The isoelectric point (pI) as determined by polyacrylamide gel isoelectric 
focusing and sucrose density gradient isoelectric focusing techniques is 
3.1-3.7, 
c) Sugar chain-constituting monosaccharides 
The following sugar chain-constituting monosaccharides have been identified 
by high-performance liquid chromatography following hydrolysis as being 
bound to each subunit: mannose, galactose, N-acetylglucosamine, 
N-acetylgalatosamine and N-acetylneuraminic acid, 
d) Circular dichroism spectrum 
The far ultraviolet CD spectrum recorded with a circular dichroism 
dispersion meter has minimum peaks at the wavelengths 208 and 222 nm, 
whereby the stimulating factor comprises an .alpha.-helix structure, 
e) Thermal stability 
The biological activity is not lost even upon heating at 60.+-.0.5.degree. 
C. for 60 minutes, and 
f) Infrared absorption spectrum as recorded for the form of a lyophilized 
powder by the transmission method using a Fouriertransform infrared 
spectrophotometer is as shown in FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION 
The colony stimulating factor (hereinafter referred to as "CSF"), which is 
glycoprotein, to be used as the essential active ingredient of the 
therapeutic agent according to the invention is described in EP-A-276 551 
(European patent application filed by one of the Assignees in the instant 
application). It has colony stimulating activity against mammalian 
monocytemacrophage series cells, and can be produced in the following 
manner. 
Urine of the healthy human origin is adjusted to pH 8.0-9.0, whereby 
viscous substances are precipitated and removed. The supernatant is 
concentrated and desalted using an ultrafiltration membrane allowing the 
passage of substances having a molecular weight of 10,000-50,000 daltons. 
After at least 200-fold concentration (at least 1% (w/v) in terms of 
protein concentration), the concentrate is adjusted to pH 6.5-7.5 and 
heat-treated at 60.degree. C. for 10 hours (for inactivation of viruses, 
etc.). The resulting precipitate is removed by centrifugation and, then 
the active component is caused to be adsorbed an anion exchanger, for 
example DEAE-cellulose. 
Said ion exchanger is washed with 0.05-0.1 M buffer (pH 6.5-7.5) and, then 
the active component is eluted with 0.2-0.4 M buffer (pH 6.5-7.5). If 
necessary, the eluate is concentrated with an ultrafiltration membrane. 
The eluate is subjected to gel filtration using a gel filtration medium, 
such as Sephacryl.RTM. S-300 (Pharmacia), equilibrated with a buffer (pH 
6.5-7.5) containing a salt, such as ammonium sulfate or sodium chloride, 
in a concentration of 1-4 M, and a fraction having the molecular weight 
range of 70,000-150,000 daltons is recovered. Then, said fraction is 
subjected to treatment for adsorption on a hydrophobic substance having 
affinity for CSF, for example on Phenyl-Sepharose.RTM. (Pharmacia). 
Elution is carried out with a buffer (pH 6.5-7.5) containing a salt in a 
concentration of 0.5-1.0 M. The eluate is concentrated with an 
ultrafiltration membrane and subjected to gel filtration using a 
high-speed liquid gel filtration column, such as TSKG-3000SW (Tosoh 
Corporation), and a fraction having the molecular weight range of 
70,000-150,000 daltons is recovered. Said fraction is again concentrated 
and subjected to treatment by adsorption on a reversed-phase 
high-performance liquid chromatography column, such as Hi-Pore.RTM. RP-304 
(Bio-Rad), equilibrated with 0.1% trifluoroacetic acid (TFA) solution (pH 
1-2). Elution is carried out with a solvent, such as acetonitrile or 
isopropyl alcohol, containing 0.1% TFA by the linear concentration 
gradient elution technique. The thus-obtained CSF is a pure substance 
having a specific activity of at least 1.times.108 units per milligram of 
protein. 
The CSF according to the present invention can also be produced by 
isolating from the culture medium of an M-CSF-producing cell line or 
M-CSF-producing cells having inserted therein DNA encoding an M-CSF gene 
by means of recombinant DNA technique. 
The thus-produced CSF according to the invention can be purified by 
utilizing the reaction of the CSF with a specific antibody. 
This production method consists of three steps, which are described one by 
one in the following. [1] Preparation of specific antibody to the colony 
stimulating glycoprotein (hereinafter referred to as "anti-CSF antibody") 
The CSF according to the invention as obtained by the above-mentioned first 
production method or a modification thereof is used to immunize mammals, 
for example rabbits, goats, sheep or horses. Thus, the CSF according to 
the invention is dissolved in physiological saline in a concentration of 
0.1-1.0 mg/ml, the solution is admixed with the equal volume of complete 
Freund's adjuvant, and the mixture is administered subcutaneously to 
mammals one or two times per week for 4-8 weeks. When the thus-immunized 
animals show an increased blood antibody titer, a booster dose is given to 
them by intravenous or subcutaneous injection and, 3-7 days thereafter, 
blood collection is performed and an antiserum to CSF is separated. The 
antibody titer of the thus-obtained antiserum against CSF is measured by 
the test to be mentioned later herein which consists in neutralization of 
the biological potency of CSF. It is desirable to select those antisera 
which have an anti-CSF antibody titer such that each milliliter thereof 
can neutralize at least 5.times.10.sup.6 units of the biological potency 
of CSF. The antiserum collected is purified by two repetitions of salting 
out with ammonium sulfate followed by DEAE-cellulose chromatography or the 
like to give the anti-CSF antibody as an immunoglobulin G or M fraction. 
If necessary, the anti-CSF antibody is further purified by applying it to 
an antigen column containing as the ligand the CSF or an impurity protein 
cross-reactive with the anti-CSF antibody as a ligand to thereby cause the 
anti-CSF antibody alone to be adsorbed on said column or by causing 
impurity proteins to be adsorbed on an appropriate column. [2] Preparation 
of antibody-bound carrier 
Any of known insoluble carriers capable of forming a chemical bond with the 
NH.sub.2 -- or COOH-- group of the antibody protein can be used as the 
insoluble carrier for binding the anti-CSF antibody. For example, there 
may be mentioned cyanogen bromide-activated or epoxidized polysaccharide 
gels, formylated polysaccharide gels, and aminoethylated or hydrazidized 
polymers. In carrying out the binding reaction between the insoluble 
carrier and the anti-CSF antibody, the antibody solution should be 
adjusted to make the same optimal for said reaction, since the optimal 
reaction conditions may vary according to the binding group of the 
insoluble carrier selected. For example, the antibody should be dissolved 
in a carbonate buffer having a pH of 8-10 in the case of cyanogen bromide 
carrier, in a solution having a pH of at least 10 in the case of 
epoxidized carriers, and in a neutral solution in the case of formylated 
carriers. The temperature conditions to be employed for said binding 
reaction also may vary depending on the insoluble carrier. Generally, 
however, the binding reaction for the practice of the invention is 
desirably carried out at a low temperature not exceeding 25.degree. C. In 
particular, in the case of cyanogen bromide-activated carriers, the 
reaction should be carried out at 4.degree. C. or below. The antibody 
quantity to be bound is generally 10-50 mg, preferably 20-30 mg, per gram 
(wet weight) of the insoluble carrier, and the antibody concentration in 
carrying out the binding reaction is adjusted to 1-4% (w/v). After 
completion of the binding reaction, those reactive groups remaining on the 
carrier without binding the antibody are inactivated by an appropriate 
treatment method, whereby the desired antibody-bound carrier is obtained. 
[3] CSF purification with antibody-bound carrier 
The antibody-bound carrier is washed with a buffer of pH 6-8 containing a 
salt, such as sodium chloride, in a concentration of 0.5-1.0 M. The 
thus-washed antibody-bound carrier is packed into a column or suspended in 
a buffer. The former is used for column chromatography and the latter for 
batchwise chromatography. The solution which contains the CSF according to 
the invention and is to be purified is, for example, a human urine 
condensate, a CSF-producing cell culture supernatant or a CSF 
gene-containing recombinant cell culture supernatant. Such solution is 
adjusted to pH 6-8 and then either equilibrated with the same buffer as 
the above-mentioned buffer used in washing the antibody-bound carrier or 
supplemented with sodium chloride to a concentration of 0.5-1.0 M. The 
thustreated solution is brought into contact with the antibody-bound 
carrier. This contact is established in the manner of column 
chromatography or batchwise chromatography. In the case of column 
chromatography, the solution is passed through the column at a temperature 
not exceeding room temperature, preferably at 10.degree. C. or below, at a 
flow rate of 5-20 ml/cm.sup.2 /hour, whereby the CSF is adsorbed on the 
antibody-bound carrier column. It is desirable that the CSF should be 
adsorbed in an amount of 500-2.times.10.sup.7 units per gram (wet weight) 
of the antibody-bound carrier. After the adsorption treatment, the 
above-mentioned buffer is passed through the column for washing and 
removing impurity substances. In the case of batchwise chromatography, the 
above treated solution is admixed with the antibody-bound carrier at a 
temperature not exceeding room temperature, preferably at 10.degree. C. or 
below, and the mixture is stirred for 1-10 hours at such temperature. 
Thereafter, the antibody-bound carrier is recovered by filtration through 
a glass filter paper or the like. Said antibody-bound carrier is 
thoroughly freed from impurity substances by washing with the 
above-mentioned buffer. The CSF specifically bound to the antibodybound 
carrier is eluted from the antibody-bound carrier with a dissociating 
solution for antigen-antibody complexes, for example acetate buffer having 
a pH of 2-3, 3-4 M thiocyanate solution or 0.1-0.2 M 2,4-dinitrophenol 
solution. In the case of column chromatography, the CSF is eluted by 
passing such eluent through the column. In the case of batchwise 
chromatography, the antibody-bound carrier is suspended in the eluent and 
the mixture is stirred, whereby the CSF is eluted. The thus-obtained CSF 
contains no impurities and is a pure form of the CSF. 
The CSF according to the invention as produced in the above manner has the 
physicochemical properties mentioned below. In testing for these 
physicohemical properties, the CSF purified by the procedure of Reference 
Example 1 was used. 
a) Molecular weight 
The molecular weight determined by sodium dodecyl sulfate-polyacrylamide 
gel electrophoresis in the absence of any reducing agent by the method of 
Laemmli (Nature, vol. 227, pages 680-685, 1970) was 70,000-90,000 daltons. 
Molecular weight determination performed by the same method but following 
reduction with 0.2 M mercaptoethanol revealed that the CSF had been 
dissociated into subunits which do not retain biological activity each 
having a molecular weight of 35,000-45,000 daltons (FIG. 1). 
FIG. 1 shows the electrophoretic pattern of the CSF according to the 
invention as revealed in sodium dodecyl sulfate-polyacrylamide gel 
electrophoresis. In FIG. 1, A to E each indicates the unreduced form 
(dimer), F and G each indicates a molecular weight marker protein, and H 
to L each indicates the reduced form (subunit). The numerical figures on 
the vertical line indicate molecular weights (.times.10.sup.3 daltons). b) 
Amino acid sequence of subunit protein 
The purified CSF was analyzed for NH.sub.2 -terminal amino acid sequence in 
the conventional manner with a vapor-phase amino acid sequencer. The 
purified CSF was then denatured with 6 M guanidine and alkylated with 
monoiodoacetic acid and, after desalting, subjected to digestion with 
trypsin, followed by decomposition with cyanogen bromide. The 
trypsin-digestion-cyanogen bromide-decomposition product. (peptide 
mixture) was fractionated by reversed-phase high-performance liquid 
chromatography using Vydac C-18. The peptide fractions separated were each 
analyzed with a vapor-phase aminoacid sequencer for determining the amino 
acid sequence of each peptide fragment. Based on the amino acid sequences 
of the respective trypsin digestion-cyanogen bromide decomposition product 
peptide fragments and the base sequence of the mRNA cloned by the present 
inventors, the primary amino acid structure of the subunit protein was 
determined. The results of sequencing are as shown in Table 1. 
The sequence from the NH.sub.2 -terminal amino acid (glutamic acid) to the 
149th amino acid (glutamic acid) is identical to that of CSF-1, which is a 
known CSF, but the sequence from the 150th to 214th-238th amino acid 
(65-89 amino acids) is quite different from that of the known CSF. 
As the COO-terminal amino acid, proline was detected as the 214th amino 
acid, and lysine as the 238th amino acid, depending on the molecular 
weight of the subunit protein. The 122nd and the 140th amino acid 
(asparagine) each has a typical N-glycoside binding structure of the 
formula Asn-X-Ser/Thr (X being an optional amino acid) and it is thought 
that these sites are the sites of sugar chain binding. 
TABLE 1 
__________________________________________________________________________ 
Subunit amino acid sequence 
__________________________________________________________________________ 
##STR1## 
ArgLeuIleAspSerGlnMetGluThrSerCysGlnIleThrPheGluPheValAspGln 
##STR2## 
MetGluAspThrMetArgPheArgAspAsnThrProAsnAlaIleAlaIleValGlnLeu 
##STR3## 
AlaCysValArgThrPheTyrGluThrProLeuGlnLeuLeuGluLysValLysAsnVal 
PheAsnGluThrLysAsnLeuLeuAspLysAspTrpAsnIlePheSerLysAsnCysAsn 
##STR4## 
TyrProLysAlaIleProSerSerAspProAlaSerValSerProHisGlnProLeuAla 
##STR5## 
##STR6## 
##STR7## 
__________________________________________________________________________ 
c) Isoelectric point 
The isoelectric point (pI) as determined by the polyacrylamide gel 
isoelectric focusing and sucrose density gradient isoelectric focusing 
techniques is 3.1-3.7. 
d) Sugar chain-constituting monosaccharides 
The constituent monosaccharides contained in the sugar chains bound to the 
polypeptide were analyzed by high-performance liquid chromatography 
following hydrolysis for liberation thereof. Aldoses and sialic acids were 
fractionated on an anion exchange column and hexosamines on a cation 
exchange column, elution being carried out by the borate buffer 
concentration gradient elution technique. The constituents were then 
subjected to post-column labelling with cyanoacetamide or arginine and 
identified by the fluorescence method. The sugar chains contained in the 
CSF molecule are variable, hence were difficult to quantitate, although 
mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine and 
N-acetylneuraminic acid were identified as constituent monosaccharides. 
e) Circular dichroism (CD) spectrum 
The CD spectrum in the far ultraviolet region was measured using a circular 
dichroism dispersion meter (JASCO model J-600) (FIG. 2). 
FIG. 2 shows the CD spectrum of the CSF according to the invention. The 
wavelength (nm) is on the horizontal axis and the ellipticity (mdeg) on 
the vertical axis. Minimum peaks are observed at the wavelengths 208 nm 
and 222 nm. It is therefore estimable that the secondary structure of the 
CSF contains an .alpha.-helix structure. 
f) Thermal stability 
The CSF was dissolved in a dilute buffer (pH 7.0) to a concentration of 1 
.mu.g/ml, and the solution was heated at 60.+-.0.5.degree. C. for 60 
minutes and then assayed for colony stimulating activity (to be mentioned 
later herein). Almost no activity decrease was observed. 
g) Infrared absorption spectrum 
The infrared absorption spectrum of the CSF in the form of a lyophilized 
powder as recorded by the transmission method (KBr window) using a 
Fourier-transform infrared spectrophotometer (Nocolet model 5DXC) was as 
shown in FIG. 3, where the horizontal line is for the wave number 
(cm.sup.-1) and the vertical line for the transmittance. 
The CSF shows strong absorption at 1650 cm.sup.-1, 1201 cm.sup.-1 and 1133 
cm.sup.-1, and medium absorption at 1537 cm.sup.-1, 1432 cm.sup.-1 and 
1068 cm.sup.-1. 
The glycoprotein having the above physicochemical properties and having 
colony stimulating activity against mammalian monocyte-macrophage series 
cells is produced from human urine by either of the above-mentioned 
production methods or obtained as otherwise disclosed herein, lyophilized 
in vials under aseptic conditions, and sealed therein in the form of a 
powder. It is also possible to add an aqueous solution containing human 
serum albumin (as CSF stabilizer) and an amino acid or sugar (as 
dissolution aid) to the CSF prior to lyophilization and subject the 
mixture to sterilization by filtration and then to lyophilization under 
aseptic conditions. 
The colony stimulating activity of the CSF according to the invention was 
determined by the method involving colony formation of mouse marrow cells 
on a single-layer soft agar gel. Thus, the CSF sample was admixed with 1 
ml of McCoy's 5A medium (GIBCO) containing 0.3% agar, 20% fetal calf serum 
(FCS) and 1.times.10.sup.5 mouse marrow cells. Incubation was carried out 
at 37.degree. C. for 7 days under a stream of 7.5% CO.sub.2 -containing 
air. Thereafter, cell aggregates consisting of 50 or more cells were 
judged as colonies and counted. The colony stimulating activity was 
expressed in units. One unit was defined as the quantity of CSF required 
for the formation of one colony. The specific activity was expressed in 
terms of the number of colonies (units) formed per milligram of the CSF 
protein. As a result, the CSF according to the invention was found to have 
a specific activity of 1.4.times.10.sup.8 units per milligram of protein. 
The colonies formed were stained with hematoxylin-eosin for morphological 
classification. It was thus found that at least 95% of the colonies formed 
were monocyte-macrophage colonies. 
The preparation according to the invention is administered, for example in 
the form of a solution in physiological saline, distilled water for 
injection or the like which has a CSF concentration of 10-100 mg/ml, by 
intravenous, intramuscular or subcutaneous injection or by intravenous 
drip. Additives such as albumin, an amino acid (e.g., glycine), sugar 
(e.g. glucose, sucrose), sugar alcohol (e.g., mannitol), an inorganic salt 
(e.g., sodium chloride) can be added to the CSF composition. 
The dose generally amounts to 1,000-150,000 units/kg body weight per 
administration once or several times per day but may suitably be increased 
or decreased depending on the symptom. 
It is recommended that the administration of said CSF be started when 
hemopoietic disorder is expected to begin after chemotherapy or 
radiotherapy. The administration may be made several times per day over 
several days (2-14 days) depending on the variation of the blood platelet 
count until said count returns to a certain constant level. 
The target of administration includes all patients with thrombocytopenia 
induced by hemopoietic disorder, without any particular limitation. 
When the preparation according to the invention was administered to such 
target patients, as described in Clinical Test Examples 1 and 2, marked 
improvements were observed in the platelet level in the circulating blood. 
No untoward side effects were observed as resulting from the 
administration. It is thus suggested that the preparation according to the 
invention should be useful as a therapeutic agent for thrombocytopenia. 
The following test example, examples and reference examples respectively 
illustrate or demonstrate the toxicity and pharmacological effects of the 
agent according to the invention and typical methods of production 
thereof. It is to be noted, however, that these examples are by no means 
limitative of the scope of the invention. 
TEST EXAMPLE 1 (TOXICITY) 
The glycoprotein prepared in Reference Example 1 was evaluated for acute 
toxicity in male C.sub.57 BL mice by the method of Richard et al. (Journal 
of Pharmacology and Experimental Therapeutics, vol. 90, page 99, 1949). 
The results obtained are shown in Table 2. 
TABLE 2 
______________________________________ 
LD.sub.50 
______________________________________ 
Intraperitoneal administration 
1 .times. 10.sup.8 units/kg (4 g/kg) 
Intravenous administration 
5 .times. 10.sup.7 units/kg (2 g/kg) 
Subcutaneous administration 
1 .times. 10.sup.8 units/kg (4 g/kg) 
______________________________________ 
EXAMPLE 1 
A malignant tumor patient was subjected to chemotherapy (first 
chemotherapy), then merely observed for a period (control phase) and, 
after a second chemotherapy, administered with 8.times.10.sup.6 units of a 
glycoprotein preparation comprising the prepared in Reference Example 1 as 
active ingredient by intravenous drip for 7 consecutive days (preparation 
administration phase). Blood granulocytes and platelets were counted at 
timed intervals. Changes in these parameters are shown in Table 3. The 
minimum platelet count and the number of days required for the platelet 
level to regain a level of at least 10.sup.5 /mm.sup.3 in the control 
phase and in the preparation administration phase are comparatively shown 
in Table 4. 
TABLE 3 
______________________________________ 
Month/day Granulocytes (/mm.sup.3) 
Platelets (/mm.sup.3) 
______________________________________ 
1/18 1,350 118,000 
1/21 1,380 115,000 
1/24 100,000 
1/27 1,020 78,000 
1/30 200 50,000 
2/1 48,000 
2/2 32 75,000 
2/4 120 142,000 
2/6 450 205,000 
2/9 1,400 330,000 
2/13 1,500 350,000 
2/16 1,770 355,000 
2/19 2,380 330,000 
2/23 2,300 320,000 
2/25 2,480 258,000 
2/27 1,720 204,000 
3/2 1,550 105,000 
3/4 1,650 100,000 
3/6 1,600 105,000 
3/9 1,630 135,000 
3/11 890 190,000 
3/13 480 270,000 
3/16 760 320,000 
3/18 1,990 300,000 
3/20 4,800 255,000 
______________________________________ 
Therapy: 
Jan. 19 CPA (Cyclophosphamide) 
600 mg 
ACR (Aclarubicin) 
60 mg 
CDDP (Cisplatinum) 
75 mg 
Feb. 23 CPA (Cyclophosphamide) 
600 mg 
ACR (Aclarubicin) 
60 mg 
CDDP (Cisplatinum) 
75 mg 
From February 24, the CSF preparation ( 8 .times. 
10.sup.6 units/day) was administered once a day for 
7 consecutive days. 
______________________________________ 
Note: 
the symbol, "--" means "not counted". 
TABLE 4 
______________________________________ 
Minimum 
Days required for 
platelet 
platelet level to 
count return to 10.sup.5 
______________________________________ 
Control phase 48,000 10 
Preparation ad- 
100,000 0 
ministration phase 
______________________________________ 
EXAMPLE 2 
Ten malignant tumor patients were subjected to a first chemotherapy, then 
observed without any treatment for a period (control phase) and, after a 
second chemotherapy, administered with 8.times.10.sup.6 units of a 
glycoprotein preparation comprising the CSF prepared in Reference Example 
1 as active ingredient by intravenous drip for 7 consecutive days 
(preparation administration phase). Blood platelets were counted at timed 
intervals. The minimum platelet count and the number of days required for 
the platelet count to regain a level of at least 10.sup.5 /mm.sup.3 in the 
control phase and in the preparation administration phase are 
comparatively shown in Table 5. 
TABLE 5 
______________________________________ 
Minimum Days required for 
platelet platelet level to 
count (.times.10.sup.3 /mm.sup.3) 
return to 10.sup.5 /mm.sup.3 
______________________________________ 
Control phase 
59.5 .+-. 30.1 
7.1 .+-. 4.8 
Preparation ad- 
119.5 .+-. 62.6 
2.5 .+-. 3.8 
ministration phase 
______________________________________ 
REFERENCE EXAMPLE 1 
Urine (200 liters) collected from healthy humans was adjusted to pH 8.5 
with a 10% sodium hydroxide solution, the resultant precipitate was 
removed by filtration, and the filtrate was concentrated and desalted with 
an ultrafiltration membrane (Amicon; H10.thrfore.50; cut-off molecular 
weight: 50,000 daltons). The concentrate was then adjusted to pH 7.0 with 
a 10% chloric acid solution and heated at 60.degree. C. in a hermetically 
closed vessel for 10 hours for sterilization. Thereafter the resultant 
precipitate was removed by centrifugation (5,000.times.g, 30 minutes), and 
the supernatant was admixed with DEAE-cellulose equilibrated with 0.02 M 
phosphate buffer (pH 7.2), for adsorption. Elution was carried out by 
treating the DEAE-cellulose with 0.02 M phosphate buffer and 0.02 
phosphate buffer (pH 7.2) supplemented with 0.05 M sodium chloride. The 
eluate was concentrated with an ultrafiltration membrane (Amicon; H1P10) 
and then subjected to gel filtration using Sephacryl S-300 (Pharmacia, 
.phi.4.times.80 cm) with 0.02 M phosphate buffer (pH 7.2) supplemented 
with 1 M ammonium sulfate. The fractions corresponding to the molecular 
weight range of 70,000-150,000 daltons as obtained in the above gel 
filtration were combined and applied to a phenyl-Sepharose 4B column 
(Pharmacia, .phi.2.times.20 cm) equilibrated with the above-mentioned 
buffer supplemented with 1 M ammonium sulfate, for adsorption. Elution was 
carried out with 0.02 M phosphate buffer (pH 7.2) supplemented with 0.5 M 
ammonium sulfate. The eluate was concentrated with an ultrafiltration 
membrane (Asahi chemical Industry, NM-3), and the concentrate was 
subjected to high-performance liquid chromatography using TSKG-3,000SW 
columns (Tosoh Corporation, .phi.4.times.600 mm.times.2) to give a 
fraction having the molecular weight range of 70,000-150,000 daltons. This 
fraction was again concentrated and subjected to highperformance liquid 
chromatography, which was performed on a reversed-phase Hi-Pore RP-304 
(Bio-Rad, .phi.4.times.150 mm) column on a linear acetonitrile 
concentration gradient (0-100%, pH 2.0). The eluent contained 0.1 M 
trifluoroacetic acid. Thus was eluted a purified CSF, which had a specific 
activity of 1.4.times.10.sup.8 units per milligram of protein. The degree 
of purification in each step of the above production process was as shown 
in Table 6. 
TABLE 6 
______________________________________ 
Purification of CSF 
Specific Re- 
Protein activity Times covery 
Purification step (*) 
(mg) (units/mg) purified 
(%) 
______________________________________ 
(1) DEAE-cellulose 
733.6 1.6 .times. 10.sup.5 
1 100 
(2) Sepnacryl S-300 
149.4 5.7 .times. 10.sup.5 
3.6 72.6 
(3) Phenyl-Sepharose 
11.3 8.8 .times. 10.sup.6 
55.0 85.4 
(4) TSKG-3,000 SW 
2.5 2.0 .times. 10.sup.7 
125.0 43.6 
(5) Hipor-RP-304 
0.25 1.4 .times. 10.sup.8 
875.0 29.9 
______________________________________ 
(*) Since untreated human urine contains a CSF-inhibiting substance, 
accurate activity assay is impossible. Therefore, the activity data are 
shown only for the DEAE-cellulose treatment step and the subsequent steps 
REFERENCE EXAMPLE 2 
From 10 rabbits immunized with the CSF obtained in Reference Example 1 and 
showing a sufficiently increased antibody titer, there was collected an 
anti-CSF antiserum, which was treated by the above-mentioned method [1] to 
give about 4 g of a purified anti-CSF antibody. The anti-CSF antibody was 
dialyzed against 0.1 M phosphate buffer (pH 7.0) and the concentration was 
adjusted to 20 mg/ml. The antibody solution (200 ml) was added to 100 g of 
formyl-Cellofine washed in advance with distilled water and with 0.1 M 
phosphate buffer, the mixture was stirred at room temperature (about 
25.degree. C.) for 2 hours, 700 mg of sodium cyanoborohydride was added, 
and the mixture was stirred for further 16 hours. Thus was prepared an 
antibody-bound carrier resulting from binding of the anti-CSF antibody to 
the formyl-Cellofine. The binding product was washed with 0.2 M 
Tris-hydrochloride buffer (pH 7.0), then 200 ml of Tris buffer containing 
500 mg of sodium cyanoborohydride was further added and the mixture was 
stirred at room temperature for 4 hours for unreacted group inactivation. 
The antibody-bound carrier was then washed thoroughly with 0.02 M 
phosphate buffer (pH 7.0) containing 0.5 M sodium chloride. Each gram of 
the antibody-bound carrier contained 29.5 mg of the anti-CSF antibody 
bound thereto. Separately, 1,000 liters of urine collected from healthy 
humans was concentrated and desalted by means of an ultrafiltration 
concentrator, treated with DEAE-cellulose for active substance adsorption 
and removal of unadsorbable impurities. Elution was carried out with 0.3 M 
sodium chloride solution, and sodium chloride was added to the eluate to a 
concentration of 0.5 M to give a CSF-containing solution. The CSF had a 
specific activity of 2 .times.10.sup.5 units/mg. This CSF-containing 
solution (total volume 500 ml) was added to 100 g of the above-mentioned 
antibody-bound carrier, and the mixture was stirred overnight (about for 
12 hours) at 10.degree. C. or below for batchwise chromatographic 
treatment. Thereafter, the antibody-bound carrier was recovered by 
filtration through a glass filter and washed thoroughly with 0.02 M 
phosphate buffer (pH 7.0) containing 0.5 M sodium chloride. After the 
washing, 500 ml of 0.2 M acetate buffer (pH 2.5) was added, and the CSF 
was eluted by stirring the mixture at 10.degree. C. for 1 hour. The eluate 
was adjusted to pH 7.0 and then concentrated and desalted with an 
ultrafiltration membrane to give about 10 mg of the CSF in a purified 
form. The purified CSF had a specific activity of 5.2.times.10.sup.7 
units/mg and a purity of at least 90% as determined by the SDS-PAGE 
method. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
aft that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.