CA1120468A - Hgi-glycoprotein capable of stimulating proliferation and differentiation of human granulocyte, process for preparing same and leukopenia curative containing same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A colony-stimulating factor having definite physical and chemical properties and a function of stimu-lating activity on human bone marrow cells to proliferate and differentiate, thereby forming granulocyte colonies, is obtained from human urine by concentrating the urine with respect to proteins contained therein by adsorption chromatography with silica gel, salting out with ammonium sulfate and other means, then removing impurities by adsorption on cation exchanger, and further purifying by ion exchanging chromatography on anion exchanger, gel filtrating chromatography with highly crosslinked gels, affinity chromatography with sugar affinitire adsorbents and electrophoresis.

Description

l This invention relates to a glycoprotein (hereinafter referred to as HGI-glycoprotein, wherein HGI
means "human granulocyte inducing".) isolated from urine of normal humans, which acts on granulopoietic stem cells in human bone marrow, thereby stimulating the prolifera~
tion and differentiation of said cells to form granulo cytes; a method for the preparation of said glycoprotein;
and curatives for leukopenia containing said glycoprotein.
Although the peripheral blood of a healthy hu~man contains 5,000 to 9,000 leukocytes per l mm3~
that of a patient of leukopenia contains below 5,000 leukocytes per l mm . Such a symptom of reduction in the ` count of leucocytes is called leukopenia. Leukopenia is associated with anomalous decrease of proliferation of bone marrow cells by some diseases, various lesions~
in bone marrow, exposure to radiation or administration : ::
of carcinostatic substances. For the therapy to leukopenia, there~have been employed chemotherapeut1cs containing glycyrrhizin or cysteine-glycine as active ingredient, ~-mercaptopropionylglycine, or Cepharantin, (a kind of alkaloi~S. These chemotherapeutics, however, are undesirable because of insufficient effectiveness and side effects. Accordingly~ many researchers have been in progress in recent years to develop therapeutical substances for treating leukopenia which are more ~

~ , ~lZ~4~8 l effective with less side effects. It was known that colony-stimulating factor (hereinafter referred to as CSF) stimulates the proliferation and differentiation of bone marrow cells. CSF acts on bone marrow cells and stimulates the proliferation and differentiation to form granulocyte or macrophage. It is an essential factor for the marrow cells, when cultured in vitro, to form gramllocytic or macrophage cell aggregates (hereinafter referred to as granulocytic or macrophage colony) by simultaneous proliferation and differentiation [Ichikawa, Y., Proceedings of the National Academy of Science, Vol~ 56, p. 488 (1966); Metcalf, D., Experi-mental Hematology, Vol. l, p. l85 (1973)]. Since CSF
induced the granulocytic and maorophage colonies from bone marrow cells, some of the researchers suggested that CSF should be regarded as separate factors that is, granulocyte inducing factor and macrophage inducing factor [Stanley~r~ E.R.f, Journal of Experimental `
Medicine, Vol. 143, p. 631 (1976)]. However, in general, these factors are assayed collectively as CSF in vitro :: :
~ ~ assay by mouse bone~ marrow cells. Many factors stimu-::
lating colony, formations in vitro by mouse bone marrow cells have been isolated from various sources i.e.
~ rln~
serum, ~ , various organ extracts, and media conditioned by various tissues and cell lines, body fluid elements such as serum and urine; conditioned media of cells such as leucocyte, and tissues ~Sheridan, J.W., Journal of whi~h Cell Physio1Ogy, Vol 78, p ~51 (1971~. CSFIacts on . .
.

6~3 l human bone marrow cells have been isolated from human origin i.e. various organ extracts~serum, media condi-tioned by tissues, ~Yff~b~X~x~e~ [Metcalf, D. and Moare, M.A.S., "Ciba Foundation Symposium 13, Haemopoietic Stem Cells", p. 157 3 E]sevier Excerpta Medica, Holland (1973)]. However, each CSF obtained from various organs, various cells and conditioned media thereof is not a single substance common to every sources. For instance, the molecular weight of CSF obtained from the media-conditioned by human placental cells is 30,000 dalton [Burgess, A.W. et al, Blood, Vol. 49, p. 573 (1977)], while that of CSF from human serum is 45,000 dalton an, S.H. et al, British Journal of Haematology, Vol.
20, p. 329 (1971) ] . Two types of CSF having molecular weights of 35,000 and less than 1,300 were isolated from media conditioned by human leukocyte CPrice~ G.B. et al~
Blood, Vol. 42, p. 341 (1973)]. Furthermore, eachCSF has different activity, some acting on either type of cells to be proliferated and differentiated to granulo-20 cyte or macropharge, others on both types of cells.Therefore3 CSF's isolated from different sources are considered to be substances different from one another ~Metcalf and Moore, loc. cit., (1973)].
It is also known that in human urine, there 25 exists a type of CSF which is capable of stimulating mouse bone marrow cells to form colonies of granulocytes and macrophages in vitro ~Stanley, E.R. et al., Federation Proceedings, Vol. 34, p. 2272 (1975); Stanly, E.R. and 13L'~4~8 1 Metcalf, D., Journal of Experimental Biology and Medical Science, ~ol. 47, p. 467 (1~69)]. It was reported that this CS~ has a molecular weight of 45,000 and stimulates the proliferation and differentiation by mouse bone marrow cells to form a macrophage dominant colony. In contrast to its stimulating effect on mouse bone marrow cells, it rarely stimulates the formation of granulo-cytic or macrophage colony by human bone marrow cells but consistently stimulates the formation of clusters.
In this specification, with respect to human bone marrow cells,-the terms "colony" and "cluster" mean cell aggregates containing 40 or more cells and 3 to less than 40 cells, respectively, in accordance with the definition of Metcalf ~Metcalf, D., Experimental Hematology~
Vol. 2, p~ 157 (1974)].
The present inventors engaged in studies on the substances having CSF activity in human urine and, as ~;~ a result, found and isolated in purified state a novel HGI-glycoprotein which, quite different from the above-said known CSF, has a molecular weight of about 8s,ooo and acts both human and mouse bone mallow cells to form pure granulocytes colonies in vitro. Further, the present inventors succeeded~in puriflcation of the H~I-glycoprotein isolated from human urine which remar-kably acts on human bone marrow cells and stimulates theproliferation and differentiation of pure granulocytes colonies (hereinafter sometimes referred to as biological activity). Further, this HGI-glycoprotein was identified, . .

preparative method thereof with good reproducibility was developed, and uses were found, leadi.ng to the accomplishment of this inventlon.
An object of this invention is to provide a novel CSF.
Another object of this invention is to provide a method for the prep-aration of this novel CS~.
A further object of this invention i.s to provide a therapeutic agent for leukopenia, which c.ontains the novel CSF.
~ ccording to this lnvention there is provided a glycoprotein from the human urine, which stimulates human bone marrow cells to form colonies of granulocytes and which has a molecular weight of 75,000 to 90,000 dalton as determined by gel flltrat;on.
The HGI-glycoprotein of the invention is produced by concentrating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the glyco-protein, eluting the glycoprotein with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel fi.ltration chroma-tography on a highly crosslinked polymer gel to develop the glycoprotein, collecting a fraction of a relative effluent of 1.11 to 1.60 to obtain the glycoprotein-containing fraction, and separating glycoprotein from the glyco-protein-containing fraction to recover the glycoprotein in pure form. The glycoprotein recovery may be effected by subjecting the collected fractions to affinity chromatography wi.th a sugar affinitive absorbent to adsorb the glyco-proteinS eluting the adsorbed glycoprotein with a 2 - 100 mM saccharide solu-tion, subjecting the eluate to preparative zone electrophoresis, eluating the glycoprotein with saline solution and recovering the glycoprotein in pure form.
The invention is described below in details.

_5_ ;

. .

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A typical procedure to prepare the ~GL-glycoprotein of this inven-tion is carriecl out in the following way. Fresh urine collected from normal h~nnans is adjustecl to pH 6-9, preferably 7-8, with dilute acid solutions or alkaline solutions and then centrifuged to remove insolubles contained in the urine. The supernatant is contacted with a silicon-containlng adsorbent such as silica gel, silica gel-magnesium silicate, diatomaceous earth, silica glass or bentonite and the adsorbed constituents were eluted with an alkaline solution of preferably pH 9 or higher. The alkaline solution which is used for the elu-tion is not specific, but is preferably an aqueous solution of ammonium hydroxide, sodium hydroxide or the like in a concentration of 0.3 to 1.5 M. The eluate thus obtained is adjusted to pH 7-8 with acid solution and added with a neutral salt such as, for example, ammonium sulfate to 70% saturation to salt out the active substance, whereby a crude protein raction containing the ~IGI-glyco-protein is obtained.

ll'~V~

1 The above crude protein fraction is re-dissolved in a small portion of an alkaline solution, freed from low molecular substances by ultrafiltration diluted with a saline buffer solution and contacted with a cation 5 exchanger (for example, carboxymethyl dextran, earboxy-methylcellulose or phosphoeellulose) to remo~e the impurities contained in this solution. The above eontaet is earried out in the eondition of neutral pH, and the erude fraetion of HGI-glycoprotein and the eation exehanger have been adjusted to pH 6-8 with preferably 0.01 - 0.15 M saline buffer solutions before the contaet.
Most of the HGI-glycoprotein passes through the eation exe~nanger without adsorption after eoneentration, the concentrated effluellt is equilibrated with a dilute buffer solution of pH 6 - ~ and applied to ion-exchange chromato-graphy with an anion exchanger, e.g. DEAE-cellulose, whieh has been ecluilibrated with the same bu~fer, the ~ HGI-glycoprotein is adsorbed onto the anion exchanger.
; Then~ the adsorbed HGI-glyeoprotein is eluted by the ~ 20 methocl of so-cal~ed linear coneentration gradient elution ; by using a 0.1 - 0.3 M saline solutions such as sodium chloride. The HGI-glyeoprotein is eluted at a salt eoncentration of 0.1 M or higher but a perfect separation is diffieult. The fraetions of effluent at 0.1 - 0.3 M
salt eoneentration are eollected and, if necessary, is sub~ected to desalting and eoncentration treatments~
It is also possible that the step-wise elution with 0.1 - 0.3 M saline solution are applied to elute the - i ;8 1 HGI ~lycoproteln from the ion exchanger.
For the purpose of further purification, the combined fraction obtained above is applied to gel filtration chromatography on a highly crosslinked polymer gel ha~ing a water regain value of 10 - 20 ml/g such as, for example, Sephadex ~ G-150 or Biogel ~ P-100 and the acti~e substances are developed with a 0.05 -0.1 M saline buffer solution. Fractions of a relative effluent volume of 1.11 to 1.60, preferably 1.11 to 1,45, are collected, desalted and concentrated or lyophilized.
The thus obtained semi purified substances containing HGI-glycoprotein can be used as pharmaceuticals.
The relative effluent volume as herein referred to is a ~olume expressed by the ratio ~e/~o (where Ve represents the volume of solvent necessary to elute the substance existing in the coIumn and Vo represents the void volume of the column).
For further purification, the semi purified substances, obtained above is dissolved in dilute saline ~ buffer solution containing 1.0 - 2.0 M such as, for example, ; a phosphate buffer solution at pH 6.o - 8.o, preferably S.O - 7.0, containing 1.0 - 2.0 M-~a~ and applied to affinity chromatography with a sugar affinitive absorbents such as, for example, concanavalin A-Sepharose 4B (supplied by Fine Chemical ~aboratory), which has been equilibrated with the same buffer solution. The HGI-glycoprotein adsorbed on affinity column is eluted : ' , :

1 with a 1.0 ~ 2.0 M saline in dilute buffer containing a 20 - 100 mM saccharides in dilute buffer solution con~aining 1.0 - 2.0 M salt at pH 6.o - 8.o, for example, saccharide is ~-methyl-D-glucoside or the li~e at pH 6.o - 8.o J preferably 6.o - 7Ø The fractions containing the HGI-glycoprotein are collected and, if necessary, desalted and concentrated or lyophilized.
For still further purification of the HGI-glycoprotein by electrophoresis, the combinedfraction obtained from affinity chromatography are applied to preparative zone electrophoresis using as s~pporting medium an acrylamide gel or agarose gel, pH 7.0 - 9.0, and the hlghly purified the HGI-glycoprotein is recovered from the supporting mediumwith a dilute saline solution under cooling conditions, desalted and concentrated or lyophLlized.
According to this invention, it is possible to recover urokinase, callicrein and lysozyme from human urine during the course of preparing HGI-glycoprotein.
The HGI-glycoprotein thus obtained is a powder which is white or faint brown in color, is taste-less, odorless and slightly hygroscopic and has the physical and chemical properties as described below.
Fig. 1 represents infrared absorption spectrum of the H~I-glycoprotein; Fig. 2 shows the correlation between the relative mobility in electrophoresis and the molecula-r weight; ~lig. 3 shows ultraviolet absorption _ 9 _ )46~3 1 spectrum of the HGI-glycoprotein; and Fig. 4 shows the relationship between the addition amount of the HCI-glycoprotein and the number of colonies developed in vitro assay.
The physical and chemical properties were determined on sample No. 6 of Example 1 (described later).
(1) Molecular weight The molecular weight of the HCI-glycoprotein of this invention was found to be about 85,ooo dalton as measured by sodium dodecyl sulfate-polyacrylamide gel elec.trophoresis and 75,000 to 90,000 dal.ton as measured by gel filtration using Sephadex ~ C-150.
Accordingly~ the most reliable molecular weight range seems to be from 75,000 to 90,000 dalton.

(2) Solubility The solubilities of` the HCI-glycoprotein in : various solvents are as shown in Table 1.
Table 1 .
: Solvent SQlubility :
Water ~ ~Soluble ~ :
Ethyl alcohol ~ Insoluble Acetone Insoluble Chloroform Slightly soluble 1 M Sodium chloride solution Soluble 10% Sucrose solution Soluble Beside, it is easily soluble in a dilute saline solution such as, for example, a dilute phosphate solution or a dilute trisaminomethane solution. It is also soluble ' ' :

: ~

~)4G~3 1 i~ a dilute saline solution in the pH range from 1 to 12.

(3) pH
The pH of a ]% aqueous solution of HGI-glyco-protein is 5.0 to 6.o~ that is, in the acidic range.

(4) Specific optical rotation.
The optical rotatlon was measured on a 0.25%
aqueous solution of HGI-gl~coprotein at 20C~ The specific optical rotation [a]20 was found to be in the range of o + l~o.

(5) Infrared absorption spectrum.
; The infrared a'osorption spectrum of HGI-glycoprotein as measured by the method of KBr pellets is as shown in Fig. 1. The characteristic absorption bands are as shwon in Table 2.

Table 2 Absorption wave Degree of Remarks . number (cm-l) ~ absorption ~ ~~

3600 - 3200 Strcng The brood absorption band seems to be origi-nated from the ~-OH
groups forming various degrees of hydrogen bands. ~ ;

1700 - 1600 Strong ~ The broad absorption band seems to be 1550 Medium ~ originatéd from ~ -CO NH- bonds of J protein fragment.
1430 - 1380 Medium 1150 - 1000 Medium The broad absgrption band seems to ~originate~t from -C-O-C-~bonds of polysaccharide fragment.

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~`-, -.

1 (6) Isoelectric point The isoelectric point of HGI-glycoprotein is pH 4.7 + 0.2, as measured by polyacrylamide gel iso-electric focussing.
t7) Color reaction Various color reactions were examined on HGI-glycoprotein dissolved in water. The results obtained are as shown in Table 3.

.
~able 3 : _ Color reactlons color F Remarks L,owry-Folin~s reaction Blue Peptide bonds Minhydrin reaction (hydrolyzed with 6N HCl at ~iolet a-amino acids 110C for 22 houra) a-Naphthol-sulfuric acid reaction (Molisch's ~iolet Saccharides reaction) . :

Indole-sulfuric acid :.
reaction (Dische's Brown "
reaction) : :

Anthrone-sulfuric acid Dark ~ "
reaction green :

Phenol-sulfuric acid Brown reaction ~
~: :

~V~6~

1 (8) Thermostability On heating a 1~ aqueous solution of HGI-glycoprotein at 60 ~ 0.5C f'or 30 minutes, the CSF
acti~rity was no more detectable.
~9) Amino acid composition o~ the protein fragment.
HGI-glycoprotein was hydrolyzed with ~
hydrochloric acid at 110C and the amino acid composition of the protein fragment was determined by means of an amino acid autoanalyzer to obtain the results as shown in Table 4.

: ~ Table 4 .

A~'n~ ~i ~ ~eight %: Mole (mM) Proline 3.2 0.392 Aspartic acid 9.8 1.038 Threonine 2.8 0.33 Serine 11.9 1.596 Glutamic acid 13.8 1.322 Glycine 11.0 ~ 2.066 Alanine 7.3 1.155 Valine ~ 6.4 0.771 Methionine 2.5 0.236 Isoleucine 2.5 0.269 Leucine : 7.0 : 0.753 Tyrosine 5.8 0.451 Phenylalanine ~ 12.8 1.050 Lysine 2.2 0.212 Histidine 1.0 0.091 Trypophan trace _ Arginine trace _ A~ 0.5 1 _ ~' ~L12~4~

1 It is seen from Table 1~ that the protein fragment of the HGI-glycoprotein is composed of 17 amino acids of which acidic and neutral amino acids dominante, while basic amino acids are minor constituents. It is also one of the characteristics that o~er 70% of the total amino acids are linear amino acids includ~ng aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine and leucine.
(10~ Electrophoresis By following the Laemuli's method ~Nature, Vol.
227, p. 680 (1970)] and using a sodium dodecyl sulfate-polyacrylamide gel, the HGI-glycoprotein which shows single band at a position of relative mobility of 0.25, trypsin inhibitor (molecular weight 21,500), ovalbumin (molecular weight 43,000), human serum albumin monomer (molecular ; weight 65, ooo ) and human serum albumin dimer (molecular weight 130~000) were simultaneously electrophored. From the mobilities o~ the substances having known molecular weights and that of the HGI-glycoprotein, the molecular weight of the latter was found to be about 85,000 (~ig. 2). In Fig.
2, a, b, c and d represent trypsin inhibitor, ovalbumin, human serum albumin monomer, and human serum albumin dimer, respectively, and the arrow represents the HGI-glycoprotein.
(11) Ultravlolet absorption spectrum.
Ultraviolet absorption spectrum of the HGI-glycoprotein, as measured on a 0.1% aqueous solution in a 1 cm silica cell, is shown in ~ig. 3. It shows the 1 maximum absorption at 280 ~ and terminal absorption in the wave length region shorter than 250 nm. The optical density ElCm at 280 ~m~ is 3.8.
(12) Sugar compositlon of polysaccharide fragment.
Neutral sugars were determined by the phenol-sulfuric acid reaction, sialic acids by the Warren's thiobarbital method [~ournal of Biological Chemistry, Vol. 234, p. 1971 (1959)], and amino sugars by the Elson-Morgan method [Biochemical Journal, Vol. 27, 10 p. 1824 (1933)]. The weight of neutral sugars were expressed in terms of glucose. The results were as follows : neutral sugars : 10.0 - 13.0%; sialic a~ids : 3.0 - 7.0%; amino sugars : less than 1.0%;
total sugar : 13.0 - 20.0%.
(13) Composition ratio of protein and polysaccharide.
The protein content of HGI-glycoprotein is 75 - 85%, as determined by the semi-micro Kieldahl method. The total sugar content is 13.0 - 20.0%, as described above.
(14) Elementary analysis The results of elementary analysis of XGI-glycoprotein are as follows : C~ 42.3 - 47.3%;
H, 5.7 - 7.8 %; N, 9.6 - 14.3 %; 0, 34-4 - 39-4 %;
S, less than 0.2%.
The HGI-glycoprotein of the above physical and chemical charactersitics has a function of stimmulat-ing the proliferation and differentiation of both human ald mouse granulocytes as seen from Test 1 (described 1 later) and shows no acute toxicity as evidenced by Test 4 (described later). ~urther, as is apparent from the resutls of Test 3 (described later), it can be utilized as leukopenia chemotherapeutics~
The HGI-glycoprotein prepared from human urine by the aforesaid procedure is aseptically lyophilized in vials and hermetically sealed. It is also possible, prior to the lyophilization~ to add to the HGI-glycoprotein an aqueous solution containing human serurn albumin as stabilizer and an amino acid or a saccharide as solubilizing aid; the resulting solution is sterilized by membrane filtration and then aseptically lyophilized.
Before using, the vial is unsealed and the HGI-glycoprotein is dissolved by adding sterilized physiological saline solution, sterile wa~er or a sterile isotonic solution.
The resulting solution is administered to the patient wlth leukopenia by intravenous, intramuscular or sub-cutaneous in~ection.
From the results of Tests 1 and 2 (described later), the effective dose is 0.75 mg or more, preferably 0.75 to 2.24 mg, per day per kg of body . weight. Semi-purified products, prepared on a large scale, having a specific biological activity of 35,000 units/mg or more such as those con~aining HGI-glycoprotein corresponding to sample No. 4 and No. 5 of Example 1 (described later) may also be used as pharmaceut~cals.
The effect of HGI-glycoprotein on the prolife-ration and differentiation of granulocytes is described

6~

1 below in detail.

Test l Stimulating effects on proliferation and dif-ferentiation of mouse and human granulocytes in vitro.
In each plastic Petri dish, 35 mm in dlameter, was plaeed 1 ml of McCoy's 5A medium containing 0.05, 0.1, 0.15 or 0.2 ~g of ~GI-glycoproteirl (sample No. 6 of Example 1), 20% of fetal calf serum, 0.3% of agar ;~ and 7.5 x ]0 mouse bone marrow eells or 25 x io:
bone marrow cells of normals or patients with iron-deficiency anemia. The medium in the Petri dish was cubated in a humidified 5% C02 atmosphere at 37C for

7 to 9 days. The difference in the number of introduced ceIls between the mouse and man was due to a greater number of committed stem cells in the ease of mouse.
:
After incubation, dlscrete eolonies eontalning more than 50 eells for mouse or more than 40 cells for human were eounted with an inverted mieroscope. For morphologic analysis of colonies,~some of them were picked up with ; 20 mierohematocrit tubes and stained wlth~0.6% orcein in 40% acetie acid. The results obtained were as shown in Fig. 4. Fig. 4 shows the interrelationship between the dose of HGI-glycoproteln and the number of colonies which were formed in vitro. In Fig. 4, -C~O-pertains to the mouse bone marrow cell and ~ to the human bone marrow cell.
As is apparent from Fig. 4, HGI-glycoprotein 6~3 1 stimmulates the proliferation and differentiation of bone marrow cells of mouse and man, thereby forming colonies and there are dose-response relationships between HGI-glycoprotein and ~ormed colony numbers.
On the morphologic analysis of the cells formed colonies, it was observed that these cells were all mature granulocytes.
As described above, IIGI-glycoprotein acts on both human and mouse bone marrow cells to ~orm colonies of granulocytes, the number of colonies being proportional to the dose of HGI-glycoprotein, and there is a definite relationship in the formation of colonies of bone marrow cells between mouse and man. Therefore, in all o~ the ~ollowing experiments, only mouse bone marrow cells were employed.

Test 2 , .
Stimu1ating effects on proliferation and differentiation of granulocyte in vivo.

Sixty C57BL male mice (20 g of average body weight) were divided at random into 6 groups of each ~h~ h S e r ~e~
10 members. One group, ~ ~Y~ as control, was subcutaneously adminstered with 0.04 mg/mouse of human serum albumin dissolved in 0.2 ml of sterile normal saline solution, once a day, for 3 consecutive days. The remaining 5 experimental groups, i.e. lst~ 2nd, 3rd, 4th and 5th group, were subcutaneously administered respectively with 0.005, 0.01, 0.02, 0.03 and 0.04 mg/mouse ~2~

1 of HGI-glycoprotein (sample No. 6 of Example 1 described later) each clissolved in 0.2 ml of sterile normal saline solution, once a day, for 3 consecutive days.
Blood samples were collected from the vena coccygea o~ each mouse before administration and 2~ 4, 6, 8 and 10 days after admlnistration. The leukocytes o~ each blood sample were stained with 1%
gentiana violet solution and leukocyte numbers were counted with a Burker-Turk counting chamber.
Further~ each blood sample was smeared on a slide glass, stained with Wright-Giemsa solution, and the proportion of granulocytes in leukocytes was rneasured ur.~er a microscope.
The number of granulocytes was calculated by the following formula:

(number of leukocytes (proportion of granulocytes in 1 mm3) x in leukocytes) = number of granulocytes in 1 mm3.

The results obtained were as~shown in Table 5.
:

' . . .

~ 46 Table 5 , \ Ir^~ 2nd ~ 3rd ¦ 4 D 0 0.005 0.01 0.02 0.03 0.04 ~ l 0 450 452 ll55450l~48 445 .

1~ 372 390 80011001ll00 1420 ._ . . ~ . _ ~, . ~

8 LllO 400 620750 980 97 ll3 410 4'~0l~60465 l~80 Note:-Each numerical value represents mean number of granulocytes per mm3 for 10 mice.

1 As shown in Table 5, it is indicated that the peripheral granulocyte counts of the experimental groups administrated with 0.01 - 0.04 mg/mouse of the HGI-glycoprotein began to increase after 2 days of administration and reached to 3 - 6 times of the count of control groups after 6 days.
The granulocyte counts decreased and returned to the normal level at 10 days. When the daily dose was increased to excess 0.04 mg, there were no significant - 20 _ 1 increase of granulocytes corresponding to increasing dose of the HGI-glycoprotein.
These results suggested that the granulo-cytosis can be sufficiently produced by daily injection of 0.01 mg or more, preferably 0.01 - 0.03 mg, of HGI-glycoprotein to a mouse (mean body weight is approximately 20 g). However, since the stimulating effect of HGI-glycoprotein on mouse bone marrow cells in vitro is an average of about 1.5 times higher than ~he effect on human bone marrow cells (Test 1), the effective dose for man is presumed to be 1.5 times as high as that for mouse determined in vivo in Test 2. Accordingly, the e~fective daily dose per kg of body weight for man is estimated as 0.75 mg or more, preferably 0.75 to 2.24 mg.
' Test 3 Protective effect of HGI-glycoprotein on leukopenia caused by carcinostatic substances.
Thirty C57BL male mice, 4 5 weeks old, were divided at random lnto 3 groups of 10 members. The control group was administered by intraperitoneal injec-tion with 30 mg/kg body weight (equivalent to 1lO LD50) of cytosine-D-arabinoside dissolved in 0.2 ml of sterile normal saline solution, once a day, for 14 consecutive days. In addition, 0.2 ml/mouse of sterile normal saline solution was subcutaneously administered once a day for 14 consecutive days. Another group (HGI-6~

1 leucoprotein administered group) was adrninistered with cytosine-~-arabinoside in the same manner as in the control group. In addition, 0.03 mg/mouse of HGI-glycoprotein (sample No. 6 of Example 1 described later) was subcutaneously administered once a day for 14 consecutive days. The remaining group (Cepharantine administered group) was administered with cytosine-D--arabinoside in the same manner as in the control group and further administered subcutaneously with 0.3 mg/

.

mouse of Cepharantine (Kaken Pharmaceuticals Co.;
conventionally employed for leukopenia) dissolved in 0.2 ml of sterile normal saline solution, once a~day for 14 consecutive days.
Blood samples were collected from the vena coccygea of each mouse before administration and 2, 4, 6, 8, 10, 12 and 14 days after administration. The number of leukocytes was measured as in Test 2 and the percentage decrease (decrement) in number of leukocytes after administration was obtained by assuming the count before administration as 100. The results were shown in Table 6.

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1 As compared with the control group, the HGI-glycoprotein administered group showed a marked preventive effect on the reduction in leucocyte count after ten days from the beginning of the HGI~glycoprotein administration, the effect being comparable or superior to that of Cepharantine. On the 14th day from the beginning of administration, the leukocyte count of the control group was reduced to 46.6%, whereas that of the HGI-glycoprotein administered group was 73.3~ the decrement beging less than that of the Cepharantine administered group. There-fore, it is presumable that the HGI-glycoprotein will be effective for the therapy of human leukopenia.
It was also confirmed that the HGI-glycoprotein is also effective when other carcinostatic substances such as, for example, 5-fluorouracil and daunomycin were administered which have been known to cause reduction in leukocyte count similarly to cytosine-D-arabinoside.
No preventive effect on the reduction in leukocyte count was observed when human serum albwnin was examined in the same manner as described above.

Test L~
Acute toxicity of the HGI-glycoprotein.
The acute toxicity of HGI-glycoprotein prepared in Example 1 (samples No. 4 and No. 6) was tested on C57BL male mice by the method of Eichied and Wilcoxon ~Journal of Pharmacology and Experimental Therapeutics, Vol 90, p. 99 (1949)]. No fatal case was found when ~l~Q~6~

1 4,000 mg/k~ bod~ weight was admi.nistered intraperito-neally or 2,000 mg/kg body weight was administered intravenously. Consequently, estimation of LD50 was practically impossible, LD50 of subcutaneous in~ection was above 4,000 mg/kg body weight and LD50 of intravenous in~ection was above 2,000 mg/kg of body weight.

Example 1 Four hundred liters of fresh urine collected from normal humans was adjustecl to pH 8 with 10% sodium hydroxide and centrifuged by menas of a continuous centrifugation at 15,000 r.p.m. at 0C to remove insolu-bles. The supernatant was ad~usted to pH 7 with 10%
hydrochloric acid and passed through a silica gel column (10 x 80 cm). The substances adsorbed or the silica gel were eluted with 40 liters of 5% ammonium solution~ The eluted~solution was adjusted to pH 7.5 with 1 N sulfuric acid, and added with ammonium sulfate to 70% saturation, and left standing at 0C for overnight. The precipitate was collected by filtration, dissolved in 2 liters of 5%
ammonlum solution, placed in cellophane tubes (Visking Co.) and diallzed against 0.05 M phosphate buffer solution (pH 6.5). The dialized solution was mess up to 10 liters with the same buffer solution and passed through the CM
Sephadex C-50 ~ ion exchange column (40 x 40 cm) which had been equilibrated with 0.05 M phosphate buffer solu-tion (pH 6.5), to adsorb the contaminants on the ion exchange resin. Ten liters of the effluent solu~ion was ~ 6~

1 concentrated by means of DlA~L0 hollow fiber ultrafiltra-tion apparatus (Amicon DC-30, U.S.A, molar weight cut off approximetly 10,000). The concentrated solution was dialized against 0.1 M tris--HCl buffer (pH 7.0) at 5C
for overnight. I'he dialized solution was made up to one liter with the same buff'er solution (the resulting solu-tion is referred to as sample No. 1).
The above solution was passed through the DEAE
cellulose column (4.0 x 40 cm) which had been equilibrated with 0.1 M tris-HCl buffer (pH 7.0) and washed the column with sufficient volume of 0.1 M tris-HCl buffer (pH 7.0).
The loaded column was carried out the step wise elution w~th 0.1 M tris-HCl buffer solution (pH 7.0) containing 0.3 M sodium chloride. The fractions capable Or effecting proliferation and differention of granulocyte, as tested in the same manner as in Test 1, were collected and dialized against 0.1 M tris-HCl buffer (pH 7.0) (this solution is referred to as sample No. 2).
The dialized solution was again passed through DEAE cellulose column (4.0 x 40 cm) which had been equi-librated with 0.1 M tris-HCl buffer (pH 7.C) and the loaded column was carried out the linear concentration gradient with elution sodium chloride (0 to 0.3 M). The active fractions were collected and added with ammonium sulfate to 70% saturation. The precipitates were collect-ed by centrifugation and dissolved in a small volume of 0.1 M tris-HCl buffer (pH 7.0) and dialized against the same buf'fer solution (this dialized solution is referred 1 to as sample No. 3~.
Twenty mllliliters of the dialized solution was applied to Sephadex G-150 column (ll.0 x 60 cm) which had been equilibrated with 0.1 M tris-HCl buffer (pH 7.0) and the effluent fractions obtained at Ve/Vo ratios of 1.11 - 1.45 were collected. The combined fraction was thoroughly dialized against distilled water at 5~ and the dialized solution was lyophilized to obtain about 500 mg of a powder (this semi-purified HGI-glycoprotein is referred to as sample No. 4).
Two hundred milligrams of the semi-purified HGI-glycoprotein was dissolved in 0.02 M phosphate buffer (pH
7.0) containing 1.0 M sodium chloride and passed through 100 ml of concanavalin A-Sepharose LIB affinity column which had been equilibrated with the same buffer. After thorough washing of the column with the same buffer, the HGI-glycoprotein was eluted with 0.02 M phosphate buf~er (pH 7.0) conbaining 50 mM ~-methyl-D-glucoside and 1.0 M
sodium chloride. The fractions which is capable of effecting proliferation and differentiation of granu-locyte in vitro were collected and dialized against distilled water. The dialized solution was lyophilozed (this is referred to as sample No. 5).
About 50 mg of the above lyophilized powder was dissolved in 1 ml of 0.125 M tris-glycine buffer (pH 6.8) containing 10% glycerine. The resulting solution was electrophored at 10 mA under cooling by means of a ~ preparative electrophoresis apparatus (Type Fuji Kabara ~lZ(~

1 II of Fuji Riken Co., Japan) employing 8% acryl.amide gel (pH 8.9; 20 mm x 25 mm). I'he fraction with a relative mobility of o.46 was recovered with 0.025 M tris-glycine buffer (pH 8.3) and was dialized against distilled water.
The dialized solution was lyophilized to obtain about 10 mg of the HGI-glycoprotein ~which is referred to as sample No. 6).
The samples No. 1 to Mo. 6 obtained in various stages of preparation were tested for the proliferation and differentiation effect on both human and mouse bone marrow cells in a manner similar to that i.n Test 1. The HGI~glycoprotein or a fraction containing same was added tc~the medium in an amount necessary to form 200 colonies per dish. The specific activlty was calculated by the following formula, wherein one unit corresponds to one colony formed:

~; Specific activity (units/mg) Number of colonies formed (units) protein content (mg) of assayed sample : The results were as shown in Table 7.

~ .

-- ~3 :::
v~ rl co 3 ~ O. , a~ ~ r l ::i r-l ~ r~ ~1ct~
r-l ~ r~ ~ ri r-l O O r ~ _ rl .~ O O O ~ O O
~ ~ ~0 ~OO O O O O
O t~ ~ ~ ~ ~ O O O
P ~ ~ ,~
~ ~ ~ L-~ o O
~ O ~ ~03t--_ 1~ 3 r-l v~.~rH ~ ~)~3 C--r~
,~ ~ ~1 ~ D
~ r~ 3 ~1 0 -=f E-l r-l Q~ ~1 00 cO
a, r~l O
O h ~ O c . O = O

~ ~ ~0 ~OO O O O O
O t~ ~j ~ O O O O
,D t~ .1~ r-l L~ r-l O
O ~1 rl ~1 0 _ M .

r-l ri~ ~ D
~3 .. . . . .
_ ZZ;ZZZZ

~ 29 -l Example 2 To lO0 mg of HGI-glycoprotein powder (sample No. 6) obtained in the same way as in Example l, was added lO0 ml of an aqueous solution containing 5% human serum albumin (Sigma Co., USA) and l~o glycine (Wako Pure Chemicals Co., Japan). The resulting solution was sterlized by Millipore filtration system (Millipore Co., USA) provided with membrane filters of 0 45 ~ pore size.
The sterilized solution was aseptically filled, in l ml portions, in vials which had been sterlized by heating at 180C for 2 hours. After lyophilization, the vials were hermetically sealed. In this way, ther were obtained 95~vials of a therapeutic agent for leukopenia, each vial containing ~ mg of H~ glycoprotein.
.
Example 3 In a same manner to that in ExampIe l, one liter o~ a concentrated solution containing HGI-glycoprotein, which was analogous to sample No. l, was prepared from l,000 liters of fresh urine collected from normal humans.
To this concentrated solution was diluted with lO
liters of O.l M tris-HCl buffer (pH 7.0). After thorough stirring, the diluted solution was reconcentrated to about one-tenth of the original volume by use of DIAFL0 ~ hollow fiber ultrafiltration apparatus. The concentrated solu-tion was added 5 liters of 0.1 M tris-HCl buffer (pH 7.0) and 5 liters of DEAE cellulose suspension containing 300 g on dry basis of DEAE cellulose, which had been equilibrated ~ 3 -6~

1 with 0.1 M tirs-~ICl buffer (pH 7.0). The mixture was stirred for 30 minutes, stand ~or 10 minutes and filtrated under vacuum on a Buchner funnel to collect the DEAE
cellulose. The collected DEAE cellulose was washed with 10 liters of 0.1 M tris-HCl buffer tpH 7.0) and recollect-ed by filtration as same as above. The DEAE cellulose was further washed with 10 liters of 0.1 M tris-HCl buffer (pH 7.0) containing 0.05 M sodium chloride and recollected by same manner as above. The DEAE cellulose thus treated, was added with 10 liters of 0.1 M tris-HCl buffer (pH 7.0)~
containing 0.3 M sodium chloride and stirred to free the HGI-glycoprotein from the DEAE cellulose. The mixture was filtrated by same manner as abo~e and filtrated solu-tion was collected. The filtrated solution was desalted by DIAFL0 hollow fiber ultrafiltration. The desalted solution was lyophilized to collect about 15 g of a powder. The powder was dissolved ln 150~ml of distilled water and applied to Sephadex G-150 column (6.o x 80 cm) which had been quilibrated with O.l M tris-HCl buffer (pH 7.0). The fractions corresponding to ~e/Vo ratios :
of 1.11 - 1.60 were collected. The combined fraction was thoroughly dialized against distilled water. The dialized solution was concentrated by DIAFL0 hollow fiher concentra-tion apparatus (Type DC-2) to about 100 ml of a concentrate containing about 3 g of crude HGI-glycoprotein. The concentrated solution was added with 1 g of glycine (Wako Pure Chemicals Co.) and 5 g of serum albumin (Sigma Co.).
The resulting solution was sterilized by fil~ration in :~ i the same manner as in E.Yample 1 and aspetically filled, in 2.5 ml portions, in vials. After aseptic lyophilization$ the vials were hermetically sealed.
Thus, there were obtained 40 vials of a therapeutic agent for leukopenia, each vial containing about 3.8 lng of the HGI-glycoprotein.

~.

~ .
. , , , : ~

Claims (6)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing a glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes, which process comprises concen-trating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the glycoprotein, eluting the glycoprotein with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly cross-linked polymer gel to develop the glycoprotein, collecting a fraction of a relative effluent volume of 1.11 to 1.60 to obtain the glycoprotein-containing fraction, and separating glycoprotein from the glycoprotein-containing fraction to recover the glycoprotein in pure form.

2. A process according to claim 1 further comprising subjecting the collected fractions of a relative effluent volume of 1.11 to 1.60 to affinity chromatography with a sugar affinitive absorbent to adsorb the glycoprotein, eluting the adsorbed glycoprotein with a 20 to 100 mM saccharide solution to obtain the glycoprotein-containing fraction.

3. A process for preparing a glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes which process comprises concen-trating human urine with respect to proteins contained therein, contacting the urinary proteins with a cation exchanger to remove impurities by adsorption on said exchanger, contacting the effluent with an anion exchanger to adsorb the glycoprotein, eluting the glycoprotein with a saline solution according to linear concentration gradient elution, subjecting the eluate to gel filtration chromatography on a highly cross-linked polymer gel to develop the glycoprotein, collecting fractions of a relative effluent of 1.11 to 1.60, subjecting the collected fractions to affinity chromatography with a sugar affinitive absorbent to adsorb the glycoprotein, eluting the adsorbed glycoprotein with a 20 - 100 mM
saccharide solution, subjecting the eluate to preparative zone electrophoresis, eluating the glycoprotein with saline solution to recover the glycoprotein in pure form.

4. A process according to claim 1 or 2 wherein the glycoprotein-contain-ing fraction has a relative effluent volume of 1.11 to 1.45.

5. A glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes whenever prepared by a process according to claim 1, 2 or 3 or by an obvious chemical equivalent thereof.

6. A glycoprotein which stimulates human bone marrow cells to form colonies of granulocytes wherein said glycoprotein has the Following physical and chemical properties:
a) solubility: soluble in water, slightly soluble in chloroform, and insoluble in ethyl alcohol and acetone;
(b) specific optical rotation: [.alpha.]20 = o + 40 (0.25% by weight aqueous solution);
(c) pH: 5.0 - 6.0 (1% by weight aqueous solution);
(d) isoelectric point: pH 4.7 + 0.2;
(e) thermostability: on being heated at 60 + 0.5°C for 30 minutes in 1% by weight aqueous solution, the stimulating function on the proliferation and differentiation of the human granulocyte is completely lost;
(f) electrophoresis: the relative mobility is 0.25 in the electro-phoresis using sodium dodecyl sulfate-polyacrylamide gel.
(g) infrared absorption: characteristic absorption at the following wave numbers (cm 1): 3600 - 3200 (strong absorption), 1700 - 1600 (strong absorption), 1550 (medium absorption), 1430 - 1380 (medium absorption), and 1150 - 1000 (broad band);
(h) color reaction: colors characteristic of saccharides are pro-duced by the .alpha.-naphthol-sulfuric acid reaction, indole-sulfuric acid reaction, anthrone-sulfuric acid reaction and phenol-sulfuric acid reaction; colors characteristic of polypeptide linkage and amino acids are produced by the Lowry Folin's reaction and by the ninhydrin reaction after hydrolysis with hydrochloric acid;
(i) constituent amino acids of the protein moiety: proline, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, triptophan and arginine;
(j) color and shape: substantially white and amorphous;
(k) sugar composition of the polysaccharide moiety: 10.0 - 13.0%
by weight in terms of glucose of neutral sugars, 3.0 - 7.0% by weight of sialic acids and less than 1% by weight of amino sugars;
(l) weight ratio of protein to polysaccharide: 75 - 85: 13.0 -20.0;
(m) elementary analysis: 42.3 - 47.3% of carbon, 5.7 - 7.8% of hydrogen, 9.6 - 14.3% of nitrogen, 34.4 - 39.4% of oxygen and less than 0.2%
of sulfur; and (n) molecular weight: 75,000 to 90,000 dalton as determined by gel filtration, whenever prepared by a process according to claim 1, 2 or 3 or by an obvious chemical equivalent thereof.