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A novel macrophage activating factor is prepared in vitro by treating glycosylated mammalian vitamin D-binding protein with glycosidases. Vitamin D-binding protein, which is isolated from blood or plasma of animals by known procedures, is thus readily converted to a highly potent macrophage activating factor.

 

U.S. Class:	435/68.1; 514/8; 530/380; 530/395; 530/402
Intern'l Class:	C12P 021/02; A61K 037/04; C07K 003/08; C07K 009/00
Field of Search:	435/68.1 514/8,2 530/350,351,380,395,402,829 424/85.1

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FIELD OF THE INVENTION

The invention relates to macrophage activation, in particular to the in vitro enzymatic production of a potent macrophage activating factor.

BACKGROUND OF THE INVENTION

A. Inflammatory Response Results in Activation of Macrophages

Microbial infections of various tissues cause inflammation which results in chemotaxis and activation of phagocytes. Inflamed tissues release lysophospholipids due to activation of phospholipase A. Inflamed cancerous tissues produce alkyl-lysophospholipids and alkylglycerols as well as lysophospholipids, because cancerous cells contain alkylphospholipids and monoalkyldiacylglyercols. These lysophospholipids and alkylglycerols, degradation products of membranous lipids in the inflamed normal and cancerous tissues, are potent macrophage activating agents (Yamamoto et al., Cancer Res. 7:2008, 1987; Yamamoto et al., Cancer Immunol. Immunother. 25:185, 1987; Yamamoto et al., Cancer Res. 24:6044, 1988).

Administration of lysophospholipids (5-20 .mu.g/mouse) and alkylglycerols (10-100 ng/mouse) to mice activates macrophages to phagocytize immunoglobulin G-coated sheep red blood cells. The macrophages phagocytize the target red blood cells via their receptors recognizing the Fc portion of the immunoglobulin G but not the C3b portion of the complement (Yamamoto et al., Cancer Res. 47:2008, 1987).

In vitro treatment of mouse peritoneal macrophages alone with lysophospholipids or alkylglycerols results in no enhanced ingestion activity (Yamamoto et al., Cancer Res. 48:6044, 1988). However, incubation of peritoneal cells (mixture of macrophages and B and T lymphocytes) with lysophospholipids or alkylglycerols for 2-3 hours produces markedly enhanced Fc-receptor-mediated phagocytic activity of macrophages (Yamamoto et al., Cancer Res. 47:2008, 1987; Yamamoto et al., Cancer Res. 48:6044, 1988).

Incubation of macrophages with lysophospholipid- or alkylglycerol-treated B and T lymphocytes in a medium containing 10% fetal calf serum developed a greatly enhanced phagocytic activity of macrophages (Yamamoto et al., Cancer Res. 48:6044, 1988; Homma and Yamamoto, Clin. Exp. Immunol. 79:307, 1990). Analysis of macrophage activating signal transmission among the nonadherent (B and T) lymphocytes has revealed that lysophospholipid- or alkylglycerol-treated B-cells can transmit a signalling factor to T-cells; in turn, the T-cells modify the factor to yield a new factor, which is capable of the ultimate activation of macrophages for ingestion capability (Yamamoto et al., Cancer Res. 48:6044, 1988).

B. Vitamin D-Binding Protein

Vitamin D-binding protein, also known as DBP, is an evolutionary conserved glycoprotein among animals (Cooke and Haddad, Endocrine Rev. 10:294 1989). DBP from animals serologically cross-reacts with human DBP (Ogata et al., Comp. Bioch. Physiol. 90B:193, 1988). Animal DBP is a genetically polymorphic plasma protein in some species and has a relative molecular weight of about 52,000. It normally constitutes about 0.5% of the plasma proteins in animals. The plasma concentration is generally about 260 .mu.g/ml. Polymorphism of the human DBP, known as "group specific component" or "Gc protein" is demonstrable by gel electrophoretic analysis, which reveals two major phenotypes: Gc1 and Gc2 (Hirschfeld et al., Nature 185:931, 1960). The entire nucleotide coding sequences of the Gc1 and Gc2 genes, and the predicted amino acid sequences, have been reported (Cooke, et al., J. Clin. Invest. 76:2420, 1985; Yang et al., Proc. Natl. Acad. Sci. USA 82:7994, 1985). Gc1 is further divided into Gc1f and Gc1s subtypes which migrate electrophoretically as two bands, "fast" and "slow", (Svasti et al., Biochem. 18:1611, 1979).

Coopenhaver et al., Arch. Biochem. Biophys. 226, 218-223 (1983) reported that a post-translational glycosylation difference occurs at a threonine residue, which appeared in a region of the protein having an amino acid difference between Gc1 and Gc2.

Viau et al., Biochem. Biophys. Res. Commun. 117, 324-331 (1983), reported a predicted structure for the O-glucosidically linked glycan of Gc1, containing a linear arrangement of sialic acid, galactose and N-acetylgalactosamine linked to a serine or threonine residue.

Polymorphism of mammalian DBP can be demonstrated by isoelectric focusing (Gahne and Juneja, Anim. Blood Grps. Biochem. Genet. 9:37, 1978; Van de Weghe et al., Comp. Biochem. Physiol. 73B:977, 1982; Ogata et al., Comp. Biochem. Physiol. 90B:193, 1988).

The animal DBP may be purified by a variety of means, which have been reported in the literature. For example, DBP may be purified by 25-hydroxyvitamin D.sub.3 -Sepharose.RTM. affinity chromatography from plasma of various animal species (Link, et al., Anal. Biochem. 157:262, 1986). DBP can also be purified by actin-agarose affinity chromatography due to its specific binding capacity to actin (Haddad et al., Biochem. J. 218:805, 1984).

Despite the characterization and intensive study of the human and animal vitamin D-binding protein, and the existence of ready methods for their purification, the conversion of these proteins to a potent macrophage activity factor has not been demonstrated until the present invention.

SUMMARY OF THE INVENTION

A process for the production of a potent macrophage activating factor is provided. Animal vitamin D-binding protein, which is an evolutionary conserved animal protein which is serologically cross-reactive with group-specific component in human serum, is a precursor of the macrophage activating factor. Animal DBP is converted to the macrophage activating factor by the action of glycosidases of B and T cells.

According to a process for preparing macrophage activating factor, animal DBP is contacted in vitro (i) with .beta.-galactosidase, or (ii) with .beta.-galactosidase in combination with sialidase, .alpha.-mannosidase or a mixture thereof. A potent macrophage activating factor is obtained in large quantities.

According to one embodiment of the invention, animal DBP, which is believed to possess an oligosaccharide moiety which includes galactose and sialic acid residues (hereinafter "DBPgs"), is contacted with .beta.-galactosidase and sialidase to provide the macrophage activating factor. According to another embodiment, DBP which is believed to possess an oligosaccharide moiety which includes galactose and .alpha.-mannose residues (hereinafter "DBPgm") is contacted with .beta.-galactosidase and .alpha.-mannosidase. In yet another embodiment, DBP which is believed to possess an oligosaccharide moiety which includes a galactose residue without sialic acid or .alpha.-mannose (hereinafter "DBPg") is contacted with .beta.-galactosidase alone to form the macrophage activating factor. Because of DBP genetic polymorphism, the macrophage activating factor is preferably prepared by contacting animal DBP with all three enzymes to obtain the macrophage activating factor, particularly when DBP purified from pooled plasma of different individuals is utilized.

The invention also relates to a macrophage activating factor prepared according to the above process or any embodiment thereof, and compositions comprising the macrophage activating factor in combination with a pharmaceutically acceptable carrier, for veterinary use.

The invention further relates to a method for inducing macrophage activation in an animal in need thereof by administering to such animal a macrophage activating effective amount of the novel macrophage activating factor.

"Animal DBP" as used herein means the genetically polymorphic animal (exclusive of human) glycoprotein, also known as "vitamin D-binding protein", including all genetic variations thereof, such as DBPg, DBPgs and DBPgm. The singular expression "DBP" is thus understood to encompass all such variants, unless stated otherwise.

By "macrophage activation" is meant the stimulation of macrophages to an increased level of phagocytic activity.

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DETAILED DESCRIPTION OF THE INVENTION

A serum factor, which has been identified as animal DBP, is converted to a macrophage activating factor by the action of B and T cell glycosidases. DBP exists as a polypeptide having attached thereto a specific oligosaccharide, portions of which are readily removable by treatment with readily available glycosidases. These glycosidases are equivalent to the functions of B and T cells upon the DBP. Upon treatment with specific glycosidases, DBP is unexpectedly converted to a highly potent macrophage activating factor. Thus, efficient conversion of DBP to the macrophage activating factor is achieved in vitro, in the absence of B- and T-cells. The novel macrophage activating factor formed by the enzymatic treatment of DBP is substantially pure and of such high potency that administration to a host of even a trace amount (500 picogram/kg of body weight) results in greatly enhanced phagocytic macrophage activity. Since the enzymatic generation of the novel factor bypasses the functions of B- and T-cells in macrophage activation, it has utility as a potent adjuvant for vaccination and as a post-infection therapeutic agent for serious infectious diseases.

T-cell lymphokine macrophage activating factor, also known as .gamma.-interferon, is generated by lymphokine-producing T-cells in small amounts, or is obtained by genetic engineering. The novel macrophage activating factor of the invention, on the other hand, may be readily obtained from DBP which can be readily purified from the plasma of animal blood according to known purification procedures.

The polymorphic DBP phenotypes are expressed inter alia as differences in the oligosaccharide attached to the polypeptide portion of the DBP molecule. The novel macrophage activating factor of the invention may be efficiently produced from animal DBP by incubation with a combination of .beta.-galactosidase and sialidase, or a combination of .beta.-galactosidase and .alpha.-mannosidase. In some instances, treatment of DBP with .beta.-galactosidase alone efficiently yields the macrophage activating factor. The in vitro conversion of DBP to macrophage activating factor by the action of commercially available enzymes is so efficient that an extremely high activity of macrophage activating factor is obtained.

Due to its genetic polymorphism in many animal species, DBP is preferably treated with all three enzymes, as an enzyme mixture. In particular, DBP obtained from pooled blood from several individuals of the species may contain more than one DBP type. Complete conversion of DBP to macrophage activating factor may thus most expeditiously be achieved by treatment with all three enzymes, as an enzyme mixture.

DBPg treated with .beta.-galactosidase alone efficiently activates macrophages. Therefore, removal of galactose from DPBg results in the formation of the macrophage activating factor. On the other hand, two glycosidases are required to convert DBP from DBPgs and DBPgm animals. Conversion of DBPgs to macrophage activity factor requires incubation with the combination of .beta.-galactosidase and sialidase. DBPgm conversion requires .beta.-galactosidase and .alpha.-mannosidase.

It is believed that animal DBP phenotypes and subtypes are characterized as glycoproteins having the following oligosaccharide structures linked to an amino acid residue of the protein portion of the molecule:

    ______________________________________
                            Representative
    DBP Type
            Oligosaccharide Animal Species
    ______________________________________
    DBPgs
             ##STR1##       monkey, bovine, sheep, goat, pig, horse
    DBPgm
             ##STR2##       bovine
    DBPg    GalGalNAc       dog, cat, rat, mouse
    ______________________________________

The glycosidases utilized in the practice of the invention are well known and commercially available. .beta.-Galactosidase, (.beta.-D-galactosidase galactohydrolase, EC 3.2.1.23) is obtained from Escherichia coli. .beta.-Galactosidase is available, for example, from Boehringer Mannheim Biochemicals, Indianapolis, Ind., cat. no. 634395.

.alpha.-Mannosidase (.alpha.-D-mannoside mannohydrolase, EC 3.2.1.24) is obtained from the jack bean (Canavalia ensiformis). It is available, for example, from Boehringer Mannheim Biochemicals, cat. no. 269611.

Sialidase, also known as "neuraminidase" (acylneuraminyl hydrolase EC 3.2.1.18), is obtained from Clostridium perfringens, Vibrio cholerae or Arthrobacter ureafaciens. All three forms of sialidase are available from Boehringer Mannheim Biochemicals, cat. nos. 107590, 1080725 and 269611.

DBP is readily converted to the macrophage activating factor by contact with a hydrolytic-effective amount of one or more of the above glycosidases. Any amount of enzyme sufficient to achieve substantially complete conversion of DBP to macrophage activating factor may be utilized. About 0.1 units (1 unit being the amount of enzyme which catalyzes 1 .mu.mole of substrate in 1 minute) of each enzyme per 1 .mu.g of DBP is more than sufficient for this purpose. Preferably, an excess of the amount of enzyme actually necessary to convert the glycoprotein to macrophage activating factor is utilized to insure complete conversion.

The DBP and enzymes may be contacted in, for example, phosphate buffer or acetate buffer. A phosphate buffer is preferred (pH 5.5). Other media known to those skilled in the art for conducting enzymatic reactions may be substituted.

The reaction may be carried out at any temperature suitable for conducting enzymatic reactions. Typically, the temperature may vary from 25.degree. C. to 37.degree. C., with about 37.degree. C. being preferred. The substrate and enzyme(s) are allowed to incubate in the reaction media until substantial conversion of DBP to macrophage activating factor is achieved. While it may be appreciated that the actual incubation times employed may depend upon several factors such as the concentration of the reactants, the reaction temperature, and the like, a reaction time of about 30 minutes at 37.degree. C. is generally sufficient to obtain complete conversion of DBP to macrophage activating factor.

Conversion of DBP to macrophage activating factor may be conducted in any vessel suitable for enzymatic reactions. It is preferred that sialidase is utilized in insoluble form, e.g., attached to beaded agarose (Sigma Chemical Co., cat. no. N-4483), to avoid contamination of the resulting macrophage activating factor with sialidase fragments of similar molecular weight. The macrophage activating factor may be produced by adding the appropriate enzyme(s) to DBP in a liquid medium, followed by subsequent filtration of the liquid to recover the macrophage activating factor. For example, the enzyme-DBP reaction mixture may be passed through a sterilized 100 kDa cut off filter (e.g. Amicon YM 100) to remove the immobilized sialidase, .beta.-galactosidase (MW=540 kDa) and .alpha.-mannosidase (MW=190 kDa). The filtrate contains substantially pure macrophage activating factor of high activity. Where the conversion of large quantities of DBP to macrophage activating factor is desired, all enzymes are most advantageously contained in the solid phase. .beta.-Galactosidase, and sialidase or .alpha.-mannosidase, most preferably a mixture of all three enzymes, is affixed to, e.g., agarose beads with a suitable coupling agent such as cyanogen bromide. Methods for attaching enzymes to solid supports are known to those skilled in the art. Conversion of DBP to macrophage activating factor by means of incubation with immobilized enzymes is preferred, as the subsequent step of separating the macrophage activating factor from the enzyme mixture is obviated.

Regardless of whether immobilized or liquid phase enzyme is utilized, it is desired to pass the product mixture through an ultrafilter, preferably a filter having a pore size no larger than about 0.45.mu., to provide an aseptic preparation of macrophage activating factor.

B-cells possess the function corresponding to .beta.-galactosidase. T-cells carry the functions corresponding to sialidase and .alpha.-mannosidase. Without wishing to be bound by any theory, it is believed that DBP is modified in vivo in an ordered sequence by the membranous enzymes of B and T lymphocytes to yield macrophage activating factor.

Activation of macrophages, which is characterized by their consequent enhanced phagocytic activity, is the first major step in a host's immune defense mechanism. Macrophage activation requires B and T lymphocyte functions, which modify DBP in a step-wise fashion, to yield the novel macrophage activating factor. Since the glycosidases used for in vitro conversion of DBP to macrophage activating factor according to the present invention correspond to the B- and the T-cell function required for production of macrophage activating factor, the in vitro enzymatic generation of the macrophage activating factor bypasses the functions of B- and T-cells. Moreover, since the herein described macrophage activating factor may be generated from blood of the same animal species undergoing treatment, side effects, such as immunogenicity, are believed to be minimal.

Following infection, microbial antigens are bound by macrophages. Most of this surface-bound antigen is internalized (i.e., phagocytized), and processed by digestion. The macrophages return some processed antigens to their surfaces so that antigenic determinants can be "presented" efficiently to antigen-specific lymphocytes. However, the binding, phagocytosis, processing and presentation of antigens requires that the macrophage first be activated. Development of the immune response following infection is thus typically delayed for 1-2 weeks, pending complete macrophage activation. This is the period during which B- and T-cells participate in generating the macrophage activating factor. During this lag period, the infection may become well-established.

I have observed the occurrence of macrophage activation in mice in less than six hours following administration of the macrophage activating factor prepared from DBP. Substantial antibody production is observed in mice in as little as 48 hours after coinjection of the macrophage activating factor and antigen. A large amount of antigen-specific antibody is produced within 96 hours. It is thus contemplated that the macrophage activating factor of the present invention, which is capable of inducing extemely rapid activation of macrophages, will be useful as an adjuvant for vaccination to enhance and accelerate the development of the immune response and to generate a large amount of antigen-specific antibodies. For the same reason, it is further contemplated that the macrophage activating factor will find utility as a post-infection therapeutic agent to accelerate antibody production, either alone or in combination with other therapeutic agents. This therapy should be particularly effective in treating infectious diseases with long incubating periods, such as rabies.

To minimize any possible immunologic reaction from administration of the macrophage activating factor, it is preferred that each animal species would receive only macrophage activating factor derived from the blood of the same species. Similarly, the risk of immunologic reaction in individual animals would be minimized by administering only the same variant of DBP-derived macrophage activating factor, in situations wherein there is intraspecies DBP polymorphism.

The macrophage activating factor may be administered to an animal to induce macrophage activation, either alone or in combination with other therapies. The amount of macrophage activating factor administered depends on a variety of factors, including the potency of the agent, the duration and degree of macrophage activation sought, the size and weight of the subject, the nature of the underlying affliction, and the like. Generally, administration of as little as about 0.5 ng of factor per kg of the subject's body weight will result in substantial macrophage activation. According to one treatment, an animal may receive about 2 ng of macrophage activating factor per kilogram of body weight every three to five days to maintain a significant level of macrophage activation.

The macrophage activating factor may be administered by any convenient means which will result in delivery to the circulation of an amount of the factor sufficient to induce substantial macrophage activation. For example, it may be delivered by intravenous or intramuscular injection. Intramuscular administration is presently preferred as the route of administration.

The macrophage activating factor may be taken up in pharmaceutically acceptable carriers, particularly those carriers suitable for delivery of proteinaceous pharmaceuticals. The factor is soluble in water or saline solution. Thus, the preferred formulation for veterinary pharmacological use comprises a saline solution of the agent. The formulation may optionally contain other agents, such as agents to maintain osmotic balance. For example, a typical carrier for injection may comprise an aqueous solution of 0.9% NaCl or phosphate buffered saline (a 0.9% NaCl aqueous solution containing 0.01M sodium phosphate, .apprxeq.pH 7.0).

The invention is illustrated by the following non-limiting examples.

EXAMPLE 1

A. Conversion of DBP to Macrophage Activating Factor

Purified DBP (1.0 .mu.g) obtained from (A) cow, (B) pooled blood of seven cows, (C) cat or (D) dog was combined with 1 ml of phosphate-buffered saline (PBS-Mg) containing 0.01M sodium phosphate, 0.9% NaCl and 1 mM MgSC.sub.4 and treated with 2 .mu.l of PBS-Mg containing 0.1 U of the enzyme combinations indicated in Table 1. The enzymes utilized were as follows:

Sialidase (Boehringer Mannheim Biochemicals, cat. no. 107590).

.alpha.-Mannosidase (Boehringer, cat. no. 107379).

.beta.-Galactosidase (Boehringer, cat. no. 634395).

The respective enzyme-DBP mixtures were incubated in microcentrifuge tubes for sixty minutes at 37.degree. C. The reaction mixture containing the enzyme-treated DBP was then diluted 10.sup.-4 in 0.1% egg albumin (EA) supplemented medium, for the following assay.

B. In Vitro Assay of Macrophage Activating-Factor

1. Preparation of Macrophage Tissue Culture

Peritoneal cells were collected by injecting 5 ml of phosphate buffered saline, containing 0.01M sodium phosphate, 0.9% NaCl and 5 units/ml heparin into the peritoneal cavity of BALB/c mice. Peritoneal cells were removed and washed by low speed centrifugation and suspended in a tissue culture medium RPMI 1640 supplemented with 0.1% egg albumin (EA medium) at a concentration of 1-2.times.10.sup.6 cells/ml. 1 ml aliquots of the cell suspension were layered onto 12 mm coverglasses which had been placed in the 16 mm diameter wells of tissue culture plates (Costar, Cambridge, Mass.). The plates were incubated at 37.degree. C. in a 5% CO.sub.2 incubator for 30 minutes to allow macrophage adherence to the coverglass. The coverglasses were removed, immersed with gentle agitation in RPMI medium to dislodge non-adherent B and T cells, and placed in fresh tissue culture wells containing EA-medium.

2. Preparation of Sheep Erythrocyte/Rabbit Anti-erythrocyte IgG Conjugates

Washed sheep erythrocytes were coated with subagglutinating dilutions of the purified IgG fraction of rabbit anti-sheep erythrocyte antibodies. A 0.5% suspension of rabbit IgG-coated sheep erythrocytes in RPMI 1640 medium was prepared for use in the following phagocytosis assay.

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3. Phagocytosis Assay.

1 ml aliquots of the diluted reaction mixture from A., above, were layered onto the macrophage-coated cover-glasses from B.1., above, and incubated for 2 hours in a 5% CO.sub.2 incubator at 37.degree. C. The culture media was then removed and 0.5 ml of the 0.5% erythrocyte-IgG conjugate suspension were added to the macrophage-coated cover-glasses and incubated for 1 hour at 37.degree. C. The coverglasses were then washed in a hypotonic solution (1/5 diluted phosphate buffered saline in water) to lyse non-ingested erythrocytes. The macrophages with ingested erythrocytes were counted. The average number of erythrocytes ingested per macrophage was also determined. Macrophage phagocytic activity was calculated as an "Ingestion index" (the percentage of macrophages which ingested erythrocytes times the average number of erythrocytes ingested per macrophage). The data are set forth in Table 1.

                  TABLE 1
    ______________________________________
    Macrophage Activation by
    Glycosidase-treated DBP
             Ingestion Index
                        B
    Glycosidases for
               A .sup.  pooled.sup.
                                 C       D
    treatment of DBP
               bovine.sup.
                        bovine.sup.
                                 cat     dog
    ______________________________________
    --         55 .+-. 10
                        67 .+-. 15
                                 77 .+-. 12
                                         73 .+-. 19
    Sialidase  59 .+-. 15
                        71 .+-. 19
                                 80 .+-. 21
                                         59 .+-. 10
    .beta.-galactosidase
               63 .+-. 18
                        76 .+-. 15
                                 278 .+-. 35
                                         284 .+-. 41
    Mannosidase
               61 .+-. 13
                        73 .+-. 28
                                 69 .+-. 15
                                         62 .+-. 26
    .beta.-galactosidase +
               295 .+-. 34
                        335 .+-. 32
                                 269 .+-. 31
                                         265 .+-. 37
    sialidase
    .alpha.-mannosidase +
               67 .+-. 22
                        54 .+-. 12
                                 73 .+-. 20
                                         67 .+-. 26
    sialidase
    .beta.-galactosidase +
               72 .+-. 15
                        188 .+-. 38
                                 266 .+-. 38
                                         252 .+-. 33
    mannosidase
    ______________________________________

It is apparent from Table 1 that bovine species display polymorphism with respect to DBP type. While the purified DBP from a single bovine individual (column A) was converted to macrophage activating factor by treatment with a combination of sialidase and .beta.-galactosidase, treatment with .beta.-galactosidase and either sialidase or .alpha.-mannosidase resulted in generation of macrophage activator from DBP purified from pooled bovine plasma of seven cows. It is thus apparent that the single bovine individual was of DBP type "gs" and that the pooled material was composed of DBP from both DBPgs and DBPgm individuals. Similarly, it is apparent from Table 1 that the cat and dog DBP donors were type DBPg, since treatment with galactosidase alone was sufficient for generation of macrophage activating factor.

The effect of macrophage activating factor concentration on activity was investigated by treating the same bovine DBPgs, pooled bovine DBP, and cat DBPg according to Example 1, at glycosidase-treated DBP dilutions of 10.sup.-4, 10.sup.-5 and 10.sup.-6 of the original 1.0 .mu.g/ml solution. The results are set forth in Table 2 (bovine DBPgs), Table 3 (pooled bovine DBP) and Table 4 (cat DBPg).

                  TABLE 2
    ______________________________________
    Macrophage activation by
    Glycosidase-treated Bovine DBPgs
                Ingestion Index
    Dilution of              Bovine DBP
    Glycosidase-  Bovine DBP treated with
    Treated       untreated  .beta.-galactosidase
    Bovine DBPgs  control    and sialidase
    ______________________________________
    10.sup.-4     63 .+-. 12 289 .+-. 11
    10.sup.-5     59 .+-. 15 322 .+-. 35
    10.sup.-6     55 .+-. 18 116 .+-. 22
    ______________________________________
              TABLE 3
    ______________________________________
    Macrophage Activation by
    Glycosidase-treated pooled bovine DBP
    Dilution of
              Ingestion Index
    Glycosidase-
              Bovine   Bovine DBP  Bovine DBP
    Treated pooled
              DBP      treated with
                                   treated with
    Bovine DBPgs
              untreated
                       .beta.-galactosidase
                                   .beta.-galactosidase
    and DBPgm control  and sialidase
                                   and .alpha.-mannosidase
    ______________________________________
    10.sup.-4 72 .+-. 25
                       312 .+-. 38 285 .+-. 38
    10.sup.-5 83 .+-. 20
                       297 .+-. 45 203 .+-. 36
    10.sup.-6 76 .+-. 18
                       145 .+-. 34 122 .+-. 23
    ______________________________________
              TABLE 4
    ______________________________________
    Macrophage Activation by
    Glycosidase-treated Cat DBPg
               Ingestion Index
    Dilution of  Cat DBP
    Glycosidase- untreated
                          Cat DBP treated with
    Cat DBPg     control  .beta.-galactosidase
    ______________________________________
    10.sup.-4    68 .+-. 26
                          320 .+-. 29
    10.sup.-5    65 .+-. 23
                          275 .+-. 23
    10.sup.-6    76 .+-. 20
                          108 .+-. 34
    ______________________________________

EXAMPLE 3

Purified DBP (1.0 .mu.g from each of the species identified in Table 5, below, was treated according to Example 1 with a mixture of .beta.-galactosidase, sialidase and .alpha.-manosidase (0.5 U each) in 1 ml of PBS-Mg containing 0.01M sodium phosphate, 0.9% NaCl and 1 mM MgSO.sub.4 for sixty minutes at 37.degree. C. The reaction mixture containing each treated DBP was then diluted 10.sup.-4 in 0.1% supplemented EA medium and assayed for macrophage activation activity according to the in vitro assay of Example 1B. The results are set forth in Table 5. It may be observed that treatment with a mixture containing all three enzymes resulted in conversion of DBP to a potent macrophage activating factor, regardless of DBP polymorphism.

                  TABLE 5
    ______________________________________
                Ingestion Index
                            Treated with
    Glycosidase-  Untreated .beta.-galactosidase +
    mannosidase   control   sialidase + .alpha.
    ______________________________________
    Monkey (Macaca fucata)
                  72 .+-. 26
                            295 .+-. 38
    Bovine (Bos taurus)
                  52 .+-. 19
                            320 .+-. 52
    Sheep (Ovis aries)
                  48 .+-. 17
                            313 .+-. 48
    Goat (Capra hircus)
                  56 .+-. 24
                            289 .+-. 32
    Pig (Sus scrofa)
                  47 .+-. 12
                            332 .+-. 27
    Horse (Equus caballus)
                  69 .+-. 23
                            266 .+-. 38
    Cat (Felis catus)
                  58 .+-. 15
                            328 .+-. 43
    Dog (Canis familigris)
                  60 .+-. 17
                            337 .+-. 18
    Rat (Fisher)  65 .+-. 25
                            284 .+-. 37
    Mouse (BALB/C)
                  71 .+-. 28
                            276 .+-. 34
    ______________________________________

DBP in 1 ml of PBS-Mg (pH 5.5) was combined with a mixture of the above-prepared agarose-immobilized enzymes (2 units each enzyme) in 1 ml of PBS-Mg (pH 5.5). The reaction mixtures were incubated in 5 ml plastic tubes at 37.degree. C. in an end-over-end mixer for 30 minutes. The reaction mixtures were thereafter spun with a table-top centrifuge at 2,000 rpm for 15 minutes. The supernatant of each reaction mixture was collected, filtered through a sterilized 0.45.mu. pore size filter (type HA, Millipore Company, Bedford, Mass.), and diluted.

B. In Vivo Assay of Macrophage Activating Factor

The enzymatically-modified DBP (100, 30, 10, 3 and 1 picogram samples) were administered intramuscularly to BALB/c mice weighing .about.20 grams. At 18 hours post-administration, peritoneal cells were collected and placed on 12 mm coverglasses in the 16 mm wells of tissue culture plates. The plates were incubated at 37.degree. C. for 30 minutes to allow adherence of macrophages. The coverglasses were washed in RPMI 1640 medium to dislodge non-adherent cells, and then placed in new wells. Rabbit IgG-coated sheep erythrocytes as prepared in Example 1B.2. were layered onto the coverglass, and a phagocytosis assay was performed as in Example 1B.3. The results are set forth in Table 6:

                  TABLE 6
    ______________________________________
    In Vivo Assay of Macrophage Activation
    by Glycosidase-treated Bovine DBPgs
    Dosage of
             Ingestion Index
    enzymatically
             Bovine DBP      Dog DBP
    modified          treated with      treated with
    DBP               .beta.-galacto-   .beta.-galacto-
    (picogram/
             untreated
                      sidase and untreated
                                        sidase and
    mouse)   control  sialidase  control
                                        sialidase
    ______________________________________
    100      63 .+-. 18
                      283 .+-. 42
                                 55 .+-. 22
                                        272 .+-. 29
    30       56 .+-. 17
                      341 .+-. 38
                                 43 .+-. 12
                                        295 .+-. 35
    10       52 .+-. 18
                      315 .+-. 44
                                 63 .+-. 17
                                        277 .+-. 41
     3       51 .+-. 12
                      141 .+-. 27
                                 51 .+-. 15
                                        128 .+-. 27
     1       65 .+-. 15
                       86 .+-. 12
                                 60 .+-. 18
                                         89 .+-. 26
    ______________________________________

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A novel, potent macrophage activating factor is prepared in vitro by treating glycosylated human group-specific component, also known as human vitamin D-binding protein, with glycosidases. Group-specific component, which is isolated from retired blood by known procedures, is thus readily converted to a highly potent macrophage activating factor.

 

U.S. Class:	435/68.1; 514/8; 530/380; 530/395; 530/402
Intern'l Class:	C12P 021/02; A61K 037/04; C07K 003/08; C07K 009/00
Field of Search:	435/68.1 514/8,2 530/350,351,380,402,829,395 424/85.1

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Viau et al., Biochem. Biophys. Res. Commun. 117, 324-331 (1983), reported a predicted structure for the O-glucosidically linked glycan of Gc1, containing a linear arrangement of sialic acid, galactose and N-acetylgalactosamine linked to a serine or threonine residue.

The Gc protein may be purified by a variety of means, which have been reported in the literature. For example, the Gc protein may be purified by 25-hydroxy-vitamin D.sub.3 -Sepharose.RTM. affinity chromatography from retired blood of the American Red Cross (Link, et al., Anal. Biochem. 157:262, 1986). The Gc protein can also be purified by actin-agarose affinity chromatography due to its specific binding capacity to actin (Haddad et al., Bioch J. 218:805, 1984).

Despite the characterization and intensive study of the human vitamin D-binding protein, and the existence of ready methods for its purification, the enzymatic conversion of this protein to a potent macrophage activity factor has not been demonstrated until the present invention.

SUMMARY OF THE INVENTION

A process for the production of a potent macrophage activating factor is provided. Human vitamin D-binding protein, which is identical to group-specific component in human serum, is a precursor of the macrophage activating factor. Group-specific component is converted to the factor by the action of glycosidases of B and T cells.

According to a process for preparing macrophage activating factor, group-specific component is contacted in vitro (i) with .beta.-galactosidase, or (ii) with .beta.-galactosidase in combination with sialidase, .alpha.-mannosidase or a mixture thereof. A potent macrophage activating factor is obtained in large quantities

According to one embodiment of the invention, group-specific component of phenotype Gc1, subtype Gc1f, is contacted with .beta.-galactosidase and sialidase to provide the macrophage activating factor. According to another embodiment, group-specific component of phenotype Gc1, subtype Gc1s, is contacted with .beta.-galactosidase and .alpha.-mannosidase. Preferably, group-specific component of phenotype Gc1, subtype Gc1s, is contacted with not only .beta.-galactosidase and .alpha.-mannosidase, but also sialidase, to ensure the conversion of the Gc1s variant (hereinafter Gc1s*) which contains sialic acid in lieu of .alpha.-mannose. Gc1s*, like Gc1f, requires treatment with .beta.-galactosidase and sialidase for conversion to macrophage activating factor. In yet another embodiment, group-specific component of phenotype Gc2 is contacted with .beta.-galactosidase alone to form the macrophage activating factor. Preferably, the macrophage activating factor is prepared by contacting pooled group-specific components comprising a mixture of Gc1f, Gc1s (Gc1s*) and Gc2 with all three enzymes to obtain the macrophage activating factor.

The invention also relates to a macrophage activating factor prepared according to the above process or any embodiment thereof, and compositions comprising the macrophage activating factor in combination with a pharmaceutically acceptable carrier.

The invention further relates to a method for inducing macrophage activation in an individual in need thereof by administering to such an individual macrophage activating effective amount of the novel macrophage activating factor.

"Group-specific component" or "Gc protein" as used herein means the genetically polymorphic glycoprotein, also known as "vitamin D-binding protein", including all genetic variations thereof, such as Gc2, Gc1, and subtypes such as Gc1f, Gc1s and Gc1s*. The singular expression "group-specific component" or "Gc protein" is thus understood to encompass all such variants, unless stated otherwise.

By "macrophage activation" is meant the stimulation of macrophages to an increased level of phagocytic activity.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1B, and 2A-2B contain the reported amino acid sequence of human group-specific component, phenotypes Gc1 and Gc2, respectively. The underlined amino acid residues at positions 152, 311, 416 and 420 differ between the two proteins.

DETAILED DESCRIPTION OF THE INVENTION

A serum factor, which has been identified as human group-specific component, is converted to a macrophage activating factor by the action of B and T cell glycosidases. Human group-specific component exists as a polypeptide having attached thereto specific oligosaccharide moieties, certain of which are readily removable by treatment with readily available glycosidases. These glycosidases are equivalent to the functions of B and T cells upon the Gc protein. Upon treatment with specific glycosidases, group-specific component is unexpectedly converted to a highly potent macrophage activating factor. Thus, efficient conversion of Gc protein to the macrophage activating factor is achieved in vitro, in the absence of intact B- and T-cells. The novel macrophage activating factor formed by the enzymatic treatment of Gc protein is substantially pure and of such high potency that administration to a host of even a trace amount (500 picogram/kg of body weight) results in greatly enhanced phagocytic macrophage activity. Since the enzymatic generation of the novel factor bypasses the functions of B-and T-cells in macrophage activation, it has utility as a therapeutic agent for inducing macrophage activation, particularly in individuals afflicted with immunodeficient diseases, cancer or other immunocompromising diseases characterized by impaired B- or T-cell function.

T-cell lymphokine macrophage activating factor, also known as .gamma.-interferon, is generated by lymphokine-producing T-cells in small amounts, or is obtained by genetic engineering. The novel macrophage activating factor of the invention, on the other hand, may be readily obtained from Gc protein which may be purified from the plasma of retired human blood in large volume, according to known purification procedures.

The human Gc protein phenotypes Gc1 and Gc2, and the Gc1 subtypes Gc1f and Gc1s, are expressed inter alia as differences in the oligosaccharides attached to the polypeptide portion of the Gc molecule. The novel macrophage activating factor of the invention may be efficiently produced from Gc1f or Gc1s protein by incubation with a combination of .beta.-galactosidase and sialidase, or a combination of .beta.-galactosidase and .alpha.-mannosidase, respectively. If the Gc1s comprises at least in part the Gc1s variant, Gc1s*, which contains sialic acid (N-acetyl-D-neuramic acid, or "NeuNAc") in lieu of .alpha.-mannose, the mixture of enzymes utilized to treat the Gc1s/Gc1s* mixture advantageously also includes sialidase. Treatment of the Gc2 protein with .beta.-galactosidase alone efficiently yields the macrophage activating factor. The in vitro conversion of Gc protein to macrophage activating factor by the action of commercially available enzymes is so efficient that an extremely high activity of macrophage activating factor is obtained.

Due to its genetic polymorphism, Gc protein obtained from pooled retired human blood will likely contain all three principal Gc types. Complete conversion of a mixture of Gc proteins to macrophage activating factor may thus most expeditiously be achieved by treatment with all three enzymes, as an enzyme mixture.

The molecules of the Gc1 and the Gc2 phenotypes are believed to differ by four amino acids at positions 152, 311, 416 and 420, as reported in the literature and reproduced in FIGS. 1A-1B and 2A-2B. The differences are as follows: ##STR1##

All three principal Gc types--Gc1s, Gc1f and Gc2--differ in the nature of the appended oligosaccharide, although it is believed that most Gc2 molecules are unglyosylated. Only the glycosylated form of Gc2 is a precursor for macrophage activating factor according to the process described herein. Incubation of each of Gc1f, Gc1s and Gc2 molecules with galactose-specific lectin beads absorbed all three macrophage activator precursor types. Thus, the outer oligosaccharide moiety of each of the three principal human Gc types is believed to be galactose.

Gc2 protein treated with .beta.-galactosidase alone efficiently activates macrophages. Therefore, removal of galactose from Gc2 protein, to the extent the molecule is present in its glycosylated form, results in the formation of the macrophage activating factor. On the other hand, two glycosidases are required to convert the Gc1 proteins to macrophage activating factor. Conversion of Gc1f to the macrophage activity factor requires incubation with the combination of .beta.-galactosidase and sialidase. Conversion of Gc1s requires .beta.-galactosidase and .alpha.-mannosidase (or .beta.-galactosidase and sialidase, in the case of Gc1s*).

The innermost sugar of the oligosaccharide moiety of Gc1 protein is N-acetylgalactosamine (Coppenhaver et al., Arch Biochem. Biophys. 226, 218-223, 1983). Treatment of Gc1 protein with endo-N-acetylglucosaminidase, which results in the cleavage of the N-acetylgalactosamine, results in a molecule which cannot be then converted to macrophage activating factor.

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It is believed that the Gc protein phenotypes and subtypes are characterized as glycoproteins having the following oligosaccharide structures linked to an amino acid residue of the protein portion of the molecule: ##STR2##

Without wishing to be bound by any theory, it is believed that the above glycosylation occurs at the specific protein portion of the Gc glycoprotein through a threonine residue occurring at amino acid position 420 (Gc1 phenotype) or through a threonine residue occurring at neighboring amino acid position 418 (Gc2 phenotype), thus forming the O-glycosidic linkage GalNAc.alpha.(1.fwdarw.0)-Thr. Thus, without wishing to be bound by any theory, the novel macrophage activating factor is believed to comprise a protein in substantially pure form having substantially the amino acid sequence of human group specific component, and a terminal N-acetylgalactosamine group linked to an amino acid residue, most likely threonine 420 and/or 418.

Further without wishing to be bound by any theory, it is believed that Gc types 1f and 1s* have the same oligosaccharide moiety, but differ in amino acid sequence. The site of the yet unknown variation in amino acid sequence is postulated to be present in the vicinity of amino acid position 420.

Human Gc protein of high purity for use in the process of the invention is most readily prepared by 25-hydroxyvitamin D.sub.3 -Sepharose.RTM. affinity chromatography from retired blood according to the procedure of Link et al., Anal. Buiochem. 157, 262 (1986), the entire disclosure of which is incorporated herein by reference. The Gc protein may also be purified by actin-agarose affinity chromatography according to the procedure of Haddad et al., Biochem. J. 218, 805 (1984), which takes advantage of the binding specificity of Gc protein for actin. The entire disclosure of Haddad et al., is incorporated herein by reference. Other methods of obtaining Gc protein in high purity are reported in the literature.

The glycosidases utilized in the practice of the invention are well known and commercially available. Galactosidase, (.beta.-D-galactosidase galactohydrolase, Ec 3.2.1.23) is obtained from Escherichia coli. .beta.-Galactosidase is available, for example, from Boehringer Mannheim Biochemicals, Indianapolis, Ind., cat. no. 634395.

.alpha.-Mannosidase (.alpha.-D-mannoside mannohydrolase, EC 3.2.1.24) is obtained from the jack bean (Canavalia ensiformis). It is available, for example, from Boehringer Mannheim Biochemicals, cat. no. 269611.

Sialidase, also known as "neuraminidase" (acylneuraminyl hydrolase EC 3.2.1.18), is obtained from Clostridium perfringens, Vibrio cholerae or Arthrobacter ureafaciens. All three forms of sialidase are available from Boehringer Mannheim Biochemicals, cat. nos. 107590, 1080725 and 269611.

Gc protein is readily converted to the macrophage activating factor by contact with a hydrolytic-effective amount of one or more of the above glycosidases. Any amount of enzyme sufficient to achieve substantially complete conversion of Gc protein to macrophage activating factor may be utilized. About 0.1 units (1 unit being the amount of enzyme which catalyzes 1 .mu.mole of substrate in 1 minute) of each enzyme per 2.6 .mu.g of Gc protein is more than sufficient for this purpose. Preferably, an excess of the amount of enzyme actually necessary to convert the glycoprotein to macrophage activating factor is utilized to insure complete conversion.

The Gc protein and enzymes may be contacted in, for example, phosphate buffer or acetate buffer. A phosphate buffer is preferred (pH 5.5). Other media known to those skilled in the art for conducting enzymatic reactions may be substituted.

The reaction may be carried out at any temperature suitable for conducting enzymatic reactions. Typically, the temperature may vary from 25.degree. C. to 37.degree. C., with about 37.degree. C. being preferred. The substrate and enzyme(s) are allowed to incubate in the reaction media until substantial conversion of the Gc protein to macrophage activating factor is achieved. While it may be appreciated that the actual incubation times employed may depend upon several factors such as the concentration of the reactants, the reaction temperature, and the like, a reaction time of about 30 minutes at 37.degree. C. is generally sufficient to obtain complete conversion of Gc protein to macrophage activating factor.

Conversion of Gc protein to macrophage activating factor may be conducted in any vessel suitable for enzymatic reactions. It is preferred that sialidase is utilized in insoluble form, e.g., attached to beaded agarose (Sigma Chemical Co., cat. no. N-4483), to avoid contamination of the resulting macrophage activating factor with sialidase fragments of similar molecular weight. The macrophage activating factor may be produced by adding the appropriate enzyme(s) to Gc protein in a liquid medium, followed by subsequent filtration of the liquid to recover the macrophage activating factor. For example, the enzyme-Gc protein reaction mixture may be passed through a sterilized 100 kDa cut off filter (e.g. Amicon YM 100) to remove the immobilized sialidase, .beta.-galactosidase (MW=540 kDa) and .alpha.-mannosidase (MW=190 kDa). The filtrate contains substantially pure macrophage activating factor of high activity.

Where the conversion of large quantities of Gc protein to macrophage activating factor is desired, all enzymes are most advantageously contained in the solid phase. .beta.-Galactosidase, and sialidase or .alpha.-mannosidase, most preferably a mixture of all three enzymes, is affixed to, e.g., agarose beads with a suitable coupling agent such as cyanogen bromide. Methods for attaching enzymes to solid supports are known to those skilled in the art. Conversion of Gc protein to macrophage activating factor by means of incubation with immobilized enzymes is preferred, as the subsequent step of separating the macrophage activating factor from the enzyme mixture is obviated.

Regardless of whether immobilized or liquid phase enzyme is utilized, it is desired to pass the product mixture through an ultrafilter, preferably a filter having a pore size no larger than about 0.45.mu., to provide an aseptic preparation of macrophage activating factor.

Without wishing to be bound by any theory, it is believed that B-cells possess the function corresponding to .beta.-galactosidase, and that T-cells carry the functions corresponding to sialidase and .alpha.-mannosidase. It is believed that Gc protein is modified in vivo in an ordered sequence by the membranous enzymes of B and T lymphocytes to yield macrophage activating factor.

Activation of macrophages, which is characterized by their consequent enhanced phagocytic activity, is the first major step in a host's immune defense mechanism. Macrophage activation requires B and T lymphocyte functions, which modify Gc protein in a step-wise fashion, to yield the novel macrophage activating factor. Since the glycosidases used for in vitro conversion of Gc protein to macrophage activating factor according to the present invention correspond to the B- and the T-cell function required for production of macrophage activating factor, the in vitro enzymatic generation of the macrophage activating factor bypasses the functions of B- and T-cells. Thus, in vitro enzymatic-generated macrophage activating factor may be used for the therapy of immuno-deficient diseases, cancer and other disease conditions characterized by the immunocompromise of the afflicted individual. Moreover, since the herein described in vitro-generated macrophage activating factor is of human origin, side effects, such as immunogenicity, are believed to be minimal.

To minimize any possible immunologic reaction from administration of the macrophage activating factor, it is preferred that individuals of phenotype Gc1 would receive only Gc1 -derived macrophage activating factor. Similarly, the risk of immunologic reaction in Gc2 individuals would be minimized by administering only Gc2-derived macrophage activating factor.

The novel macrophage activating factor is also believed useful in the treatment of disorders characterized by a disruption or loss of B- or T-cell function. Such disorders may be characterized by a lack of macrophage activation. Addition of exogenous macrophage activating factor of the invention will result in the restoration of macrophage activity, even in the absence of complete B- or T-cell function.

The macrophage activating factor may be administered to an individual to induce macrophage activation, either alone or in combination with other therapies. The amount of macrophage activating factor administered depends on a variety of factors, including the potency of the agent, the duration and degree of macrophage activation sought, the size and weight of the subject, the nature of the underlying affliction, and the like. Generally, administration of as little as about 0.5 ng of factor per kg of the subject's body weight will result in substantial macrophage activation. According to one treatment, a human subject may receive as little as about 30-35 ng of macrophage activating factor every three to five days to maintain a significant level of macrophage activation.

The macrophage activating factor may be administered by any convenient means which will result in delivery to the circulation of an amount of the factor sufficient to induce substantial macrophage activation. For example, it may be delivered by intravenous or intramuscular injection. Intravenous administration is presently preferred as the route of administration.

The macrophage activating factor may be taken up in pharmaceutically acceptable carriers, particularly those carriers suitable for delivery of proteinaceous pharmaceuticals. The factor is soluble in water or saline solution. Thus, the preferred formulation for pharmacological use comprises a saline solution of the agent. The formulation may optionally contain other agents, such as adjuvants to maintain osmotic balance. For example, a typical carrier for injection may comprise an aqueous solution of 0.9% NaCl or phosphate buffered saline (a 0.9% NaCl aqueous solution containing 0.01M sodium phosphate, .apprxeq.pH 7.0).

The invention is illustrated by the following non-limiting examples.

EXAMPLE 1

A. Conversion of Gc Protein to Macrophage Activating Factor

Gc protein (2.6 .mu.g; Gc1 or Gc2) in 1 ml of phosphate-buffered saline (PBS-Mg) containing 0.01M sodium phosphate, 0.9% NaCl and 1 mM MgSO.sub.4 was treated with 2 .mu.l of PBS-Mg containing 0.1 U of the following enzymes or enzyme combinations.

Gc1f/Gc1s* conversion: sialidase (Boehringer Mannheim Biochemicals, cat. no. 107590) and .beta.-galactosidase (Boehringer, cat. no. 634395);

Gc1s conversion: .alpha.-mannosidase (Boehringer, cat. no. 107379) and .beta.-galactosidase;

Gc2 conversion: .beta.-galactosidase only.

The respective enzyme-Gc protein mixtures were incubated in microcentrifuge tubes for thirty minutes at 37.degree. C. The reaction mixture containing the treated Gc protein was then diluted 10.sup.-4, 10.sup.-5 or 10.sup.-6 in 0.1% egg albumin (EA) medium, for the following assay.

B. In Vitro Assay of Macrophage Activating Factor

1. Preparation of Macrophage Tissue Culture

Peritoneal cells were collected by injecting 5 ml of phosphate buffered saline, containing 0.01M sodium phosphate, 0.9% NaCl and 5 units/ml heparin into the peritoneal cavity of BALB/c mice. Peritoneal cells were removed and washed by low speed centrifugation and suspended in a tissue culture medium RPMI 1640 supplemented with 0.1% egg albumin (EA) medium at a concentration of 1-2.times.10.sup.6 cells/ ml. 1 ml aliquots of the cell suspension were layered onto 12 mm coverglasses which had been placed in the 16 mm diameter wells of tissue culture plates (Costar, Cambridge, Mass.). The plates were incubated at 37.degree. C. in a 5% CO.sub.2 incubator for 30 minutes to allow macrophage adherence to the coverglass. The coverglasses were removed, immersed with gentle agitation in RPMI medium to dislodge non-adherent B and T cells, and placed in fresh tissue culture wells containing EA-medium.

2. Preparation of Sheet Erythrocyte/Rabbit Anti-erythrocyte IgG Conjugates

Washed sheep erythrocytes were coated with subagglutinating dilutions of the purified IgG fraction of rabbit anti-sheep erythrocyte antibodies. A 0.5% suspension of rabbit IgG-coated sheep erythrocytes in RPMI 1640 medium was prepared for use in the following phagocytosis assay.

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3. Phagocytosis Assay

1 ml aliquots of the diluted reaction mixture from A., above, were layered onto the macrophage-coated coverglasses from B.1., above, and incubated for 2 hours in a 5% CO.sub.2 incubator at 37.degree. C. The culture media was then removed and 0.5 ml of the 0.5% erythrocyte-IgG conjugate suspension were added to the macrophage-coated coverglasses and incubated for 1 hour at 37.degree. C. The coverglasses were then washed in a hypotonic solution (1/5 diluted phosphate buffered saline in water) to lyse non-ingested erythrocytes. The macrophages with ingested erythrocytes were counted. The average number of erythrocytes ingested per macrophage was also determined. Macrophage phagocytic activity was calculated as an "Ingestion index" (the percentage of macrophages which ingested erythrocytes.times.the average number of erythrocytes ingested per macrophage). The data is set forth in Table 1 (Gc1 ) and Table 2 (Gc2).

                  TABLE 1
    ______________________________________
    Dilution of
             Ingestion Index
    Glycosidase-
             Gc1      Gc1 treated with
                                   Gc1 treated with
    Treated  untreated
                      .beta.-galactosidase
                                   .beta.-galactosidase
    Gc1.sup.1 Protein
             control  and sialidase
                                   and .alpha.-mannosidase
    ______________________________________
    10.sup.-4
             75 .+-. 10
                      352 .+-. 15  295 .+-. 11
    10.sup.-5
             82 .+-. 11
                      286 .+-. 11  210 .+-. 8
    10.sup.-6
             79 .+-. 8
                      122 .+-. 7   109 .+-. 13
    ______________________________________
     .sup.1 Mixture of Gc1f and Gc1s
              TABLE 2
    ______________________________________
    Dilution of    Ingestion Index
    Glycosidase-   Gc2
    Treated        untreated
                            Gc2 treated with
    Gc2 Protein    control  .beta.-galactosidase
    ______________________________________
    10.sup.-4      65 .+-. 13
                            325 .+-. 16
    10.sup.-5      69 .+-. 11
                            208 .+-. 17
    10.sup.-6      71 .+-. 20
                            116 .+-. 5
    ______________________________________

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2. Conversion of Gc Protein to Macrophage Activating Factor

Gc protein (2.6 .mu.g; Gc1, Gc2, or mixture thereof) in 1 ml of PBS-Mg (pH 5.5) was combined with a mixture of the above-prepared agarose-immobilized enzymes (2 units each enzyme) in 1 ml of PBS-Mg (pH 5.5). The reaction mixtures were incubated in 5 ml plastic tubes at 37.degree. C. in an end-over-end mixer for 30 minutes. The reaction mixtures were thereafter spun with a table-top centrifuge at 2,000 rpm for 15 minutes. The supernatant of each reaction mixture was collected, filtered through a sterilized 0.45.mu. pore size filter (type HA, Millipore Company, Bedford, Mass.), and diluted.

B. In Vivo Assay of Macrophage Activating Factor

The enzymatically-modified Gc protein (40, 10, 4 and 1 picogram samples) were administered intramuscularly to BALB/c mice weighing .about.20 grams. At 18 hours post-administration, peritoneal cells were collected and placed on 12 mm coverglasses in the 16 mm wells of tissue culture plates. The plates were incubated at 37.degree. C. for 30 minutes to allow adherence of macrophages. The coverglasses were washed in RPMI 1640 medium to dislodge non-adherent cells, and then placed in new wells. Rabbit IgG-coated sheep erythrocytes as prepared in Example 1B.2. were layered onto the coverglass, and a phagocytosis assay was performed as in Example 1B.3. The results are set forth in Table 3:

                  TABLE 3
    ______________________________________
    Dosage of enzyma-
                Ingestion Index
    tically modified
                Untreated Control
                              Glycosidase-treated
    Gc protein           Gc1 +           Gc1 +
    (picogram/mouse)
                Gc1      Gc2      Gc1    Gc2
    ______________________________________
    40          57 .+-. 16
                         59 .+-. 7
                                  322 .+-. 19
                                         314 .+-. 11
    10          55 .+-. 10
                          63 .+-. 13
                                  353 .+-. 16
                                         332 .+-. 14
     4          51 .+-. 12
                         45 .+-. 8
                                  163 .+-. 18
                                         152 .+-. 13
     1          63 .+-. 18
                         56 .+-. 9
                                  114 .+-. 3
                                         106 .+-. 5
    ______________________________________

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( 1 of 1 )

The present invention relates to an improved process for the preparation of 1-desoxynojirimycin. 1-Desoxynojirimycin can be reacted by alkylation on the nitrogen atom to give various saccharase inhibitors which are used therapeutically in the treatment of diabetes mellitus.

 

U.S. Class:	435/84; 435/85; 435/228; 435/229; 435/230
Intern'l Class:	C12P 019/26; C12P 019/28; C12N 009/80; C07H 005/06
Field of Search:	435/84,85,230,228,229

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The present invention relates to an improved process for the preparation of 1-desoxynojirimycin. 1-Desoxynojirimycin can be reacted by alkylation on the nitrogen atom to give various saccharase inhibitors which are used therapeutically in the treatment of diabetes mellitus (W. Puls, U. Keup, H.P. Krause, G. Thomas, and F. Hoffmeister, Naturwissenschaften, 64 (1977) 536 et seq.).

1-Desoxynojirimycin is prepared by a combined chemical-microbiological process (EP 49,858).

An important intermediate in the preparation of 1-desoxynojirimycin is aminosorbose, which, however, is unstable. To remove the N-acetyl and N-formyl protecting group, either extremely acidic or extremely alkaline conditions have to be selected. Above pH 2 and below pH 13, virtually no removal of the acyl group takes place. Under the abovementioned pH conditions, however, considerable desoxyno3irimycin losses are observed. These result on the one hand from cyclisation reactions of the aminosorbose itself to give, for example, hydroxylated pyridines, on the other hand, coloured secondary components which have not been specified in more detail are formed. Thus, on acidic removal of the acyl group only a yield of about 27 % of desoxynojirimycin was observed and on alkaline removal a yield of about 60 % of desoxynojirimycin was observed.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that in the range from 8 to 9.5, the removal of an N-phenacetyl protecting group by means of the known enzyme penicillin acylase (penicillin amidohydrolase (EC 3.5.1.11)) isolated from Escherichia coli can be achieved with substantially better yield and purity of 1-desoxynojirimycin than with chemical methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing the yield of 1-desoxynojirinycin over time when prepared by chemical methods and enzymatic methods.

DETAILED DESCRIPTION OF THE INVENTION

The reaction mentioned is described by the following reaction equations: ##STR1##

At pH 13, in contrast to pH 8-9, a very rapid decomposition of aminosorbose is observed, which leads to the loss of 40-50 % of 1-desoxynojirimycin.

In the enzymatic removal of the N-phenacetyl protecting group using penicillin acylase, however, this rapid decomposition is lacking, so that the yield of 1-desoxynojirimycin can be increased. The yield can be still further increased by an increase in the enzyme concentration and thus a reduction of the cleavage time (FIG. 1).

Penicillin acylase has hitherto only been described for the removal of the N-phenacetyl group on penicillin and cephalosporin parent substances, amino acids and peptides (J.G. Shewale, B.S. Deshphande, V.K. Sudhakaran and S.S. Ambedkar, Process Biochemistry, June 1990, 97-103).

The removal of the phenacetyl group from amino sugars using penicillin acylase is new and also utilisable for other unstable N-acyl-aminopentoses and aminohexoses.

In the process described, purified free and carrier-bound penicillin acylase, for example, can be employed.

EXAMPLE 1

Use of soluble penicillin acylase

3 kg of N-phenacetyl-aminosorbitol are dissolved in 150 litres of water and the pH of the solution is adjusted to about 4.5 and kept at this pH. The solution is warmed to 37.degree. C. and about 6 kg of centrifuged bacterial cells (Gluconobacter suboxydans) are added and the suspension is aerated intensively with air whilst stirring for 4 hours.

In this period, the N-phenacetyl-aminosorbitol is quantitatively oxidised to N-phenacetyl-aminosorbose microbiologically. The Gluconobacter cells are completely removed from the suspension by centrifugation, the clear supernatant is adjusted to pH 9 using sodium hydroxide solution and 2.7 million units of penicillin acylase are added (isolation of the enzyme and definition of the unit are described in the literature reference: C. Kutzbach and E. Rauenbusch, Hoppe-Seyler's Z. Physiol.-Chem., 354 (1974) 45-53). The removal of the phenylacetic acid is monitored by means of high pressure liquid chromatography via the determination of the liberated phenylacetic acid and/or the decrease in the N-phenacetylaminosorbose. After a reaction time of about 45-60 minutes, the removal of protecting groups is complete and 270 g of sodium borohydride are added. Reduction and cyclisation to give 1-desoxynojirimycin last 60 minutes. The excess of sodium borohydride is then destroyed by addition of acetone or HC1.

Further working-up to give pure 1-desoxynojirimycin is carried out by chromatography via ion exchangers and subsequent crystallisation as described in EP 49,859 and in the literature reference: Y. Eze, S. Maruo, K. Miyazaki and M. Kawamata, Agric. Biol. Chem., 49 (1985) 1119-1125.

After crystallisation, about 1.3 kg of 1-desoxynojirimycin (about 80 % molar yield) having a purity by HPLC >95 % are obtained.

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EXAMPLE 2

Use of carrier-bound penicillin acylase Carrying-out takes place as described in Example 1. To remove the N-phenacetyl group, 5.0 million units of penicillin acylase resin prepared according to DOS (German Offenlegungsschrift) 2,215,687 are added. Before the isolation of 1-desoxynojirimycin, the enzyme resin is filtered off and washed.

The yield of 1-desoxynojirimycin is 1.4 kg (about 83 % molar yield) having a purity of HPLC>95%.

    ______________________________________
    High pressure liquid chromatography
    ______________________________________
    Column:
    LiChrospher RP 18.5 .mu.m
    (Merck, Darmstadt);
    250 mm .times. 4 mm
    Eluent:
    A: 0.05 M phosphate, pH 6.5
    B:90% acetonitrile: 10% water
    Gradient:
    Time in min.   % A    % B
     0             100    0
     2             100    0
    10              0     100
    11.5            0     100
    11.6           100    0
    16.5           100    0
    Flow rate: 1.5 ml/min.
    Temperature: 25.degree. C.
    Wavelength: 210 nm
    Injection volume: 20 .mu.L
    Retention times in minutes:
    N-phenacetyl-aminosorbitol:
                    7.1
    N-phenacetyl-aminosorbose:
                    8.7
    Phenylacetic acid:
                    4.7
    Desoxynojirimycin:
                    0.9
    ______________________________________

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( 1 of 1 )

A novel macrophage activating factor is prepared in vitro by treating glycosated vitamin D-binding protein with glycosidases. Vitamin D-binding protein, which is isolated from blood or plasma of mammals by known procedures, is thus readily converted to a highly potent macrophage activating factor.

 

U.S. Class:	514/8; 530/362; 530/380; 530/395
Intern'l Class:	A61K 037/02; C07K 007/10; C07K 009/00; C07K 015/14
Field of Search:	514/8 530/380,395,362

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