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United States Patent 5,177,001
Yamamoto January 5, 1993

In vitro enzymatic conversion of glycosylated mammalian vitamin D-binding protein to a potent macrophage activating factor

Abstract

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.


Inventors: Yamamoto; Nobuto (1040 66th Ave., Philadelphia, PA 19126)
Assignee: Yamamoto; Nobuto (Philadelphia, PA)
Appl. No.: 767742
Filed: September 30, 1991

 
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


References Cited [Referenced By]

U.S. Patent Documents

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Other References

Yamamoto et al., Cancer Res. 47:2008, 1987.
Yamamoto et al., Cancer Immunol. Immunother. 25:185, 1987.
Yamamoto et al., Cancer Res. 42:6044, 1988.
Ngwenya et al., Abstracts of the Annual Meeting of the American Society of Microbiology, Abs. E-72, p. 121 (1988).
Homma, Abstracts of the Annual Meeting of the Am. Society of Microbiology, Abs. E-74, p. 121, (1988).
Cooke et al., J. Clin. Invest. 76:2420-2424 (1985).
Yang et al., Proc. Natl. Acad. Sci. 82:7994-7998 (1985).
Haddad et al., Biochem. J. 218: 805-810, 1984.
Link et al., Analyt. Biochem. 157: 262-269, 1986.
Cooke et al., Endocrine Reviews 10: 294-307, 1989.
Ogata et al., Comp. Biochem. Physiol. 90 B; 193-199, 1988.
Gahne et al., Anim. Blood Grps. Biochem. Genet. 9: 37-40, 1978.
Van De Weghe et al., Comp. Biochem. Physiol. 73 B: 977-982, 1982.
Van Baelen et al., J. Biol. Chem. 253: 6344-6345, Sep. 25, 1978.
Svasti et al., J. Biol. Chem. 253: 4188-4194, Jun. 25, 1978.
Shinomiya et al., J. Biochem. 92: 1163-1171, 1982.

Primary Examiner: Wax; Robert A.
Assistant Examiner: Walsh; Stephen
Attorney, Agent or Firm: Seidel, Gonda, Lavorgna & Monaco

Parent Case Text



This is a continuation-in-part of copending application Ser. No. 07/576,248, filed Aug. 31, 1990, which is a continuation in part of copending application Ser. No. 07/439,223, filed Nov. 20, 1989 now abandoned. The entire disclosure of application Ser. No. 07/576,248 is incorporated herein by reference.
Claims



1. A process for producing a macrophage activating factor comprising contacting glycosylated mammalian vitamin D-binding protein in vitro with

.beta.-galactosidase, or

.beta.-galactosidase in combination with sialidase, .alpha.-mannosidase, or a mixture thereof,

and isolating the macrophage activating factor.

2. A process according to claim 1 wherein the vitamin D-binding protein is contacted with .beta.-galactosidase and sialidase.

3. A process according to claim 1 wherein the vitamin D-binding protein is contacted with .beta.-galactosidase and .alpha.-mannosidase.

4. A process according to claim 1 wherein the vitamin D-binding protein is contacted with .beta.-galactosidase.

5. A process according to claim 1 wherein the vitamin D-binding protein is contacted with a mixture of glycosidases comprising .beta.-galactosidase, sialidase and .alpha.-mannosidase.

6. A process according to claim 1 wherein the vitamin D-binding protein comprises bovine vitamin D-binding protein.

7. A process according to claim 1 wherein the vitamin D-binding protein comprises horse vitamin D-binding protein.

8. A process according to claim 1 wherein the vitamin D-binding protein comprises sheep vitamin D-binding protein.

9. A process according to claim 1 wherein the vitamin D-binding protein comprises pig vitamin D-binding protein.

10. A process according to claim 1 wherein the vitamin D-binding protein comprises goat vitamin D-binding protein.

11. A process according to claim 1 wherein the vitamin D-binding protein comprises dog vitamin D-binding protein.

12. A process according to claim 1 wherein the vitamin D-binding protein comprises cat vitamin D-binding protein.

13. A process according to claim 1 wherein the enzyme or enzymes is immobilized on a solid support.

14. A process according to claim 13 wherein the solid support comprises agarose.

15. A macrophage activating factor prepared by the process of claim 1.

16. A macrophage activating factor prepared by the process of claim 2.

17. A macrophage activating factor prepared by the process of claim 3.

18. A macrophage activating factor prepared by the process of claim 4.

19. A macrophage activating factor prepared by the process of claim 5.

20. A macrophage activating factor prepared by the process of claim 6.

21. A macrophage activating factor prepared by the process of claim 7.

22. A macrophage activating factor prepared by the process of claim 8.

23. A macrophage activating factor prepared by the process of claim 9.

24. A macrophage activating factor prepared by the process of claim 10.

25. A macrophage activating factor prepared by the process of claim 11.

26. A macrophage activating factor prepared by the process of claim 12.

27. A macrophage activating factor prepared by the process of claim 13.

28. A macrophage activating composition comprising, in combination with a pharmaceutically acceptable carrier, a macrophage activating factor formed by treating glycosylated vitamin D-binding protein in vitro with

.beta.-galactosidase, or

.beta.-galactosidase in combination with sialidase, .alpha.-mannosidase, or mixtures thereof.

29. A macrophage activating composition according to claim 28 wherein the vitamin D-binding protein is treated with .beta.-galactosidase and sialidase.

30. A macrophage activating composition according to claim 28 wherein the vitamin D-binding protein is treated with .beta.-galactosidase and .alpha.-mannosidase.

31. A macrophage activating composition according to claim 28 wherein the vitamin D-binding protein is treated with .beta.-galactosidase.

32. A macrophage activating composition according to claim 28 wherein the vitamin D-binding protein is treated with a mixture of glycosidases comprising .beta.-galactosidase, sialidase and .alpha.-mannosidase.

33. A method for inducing macrophage activation in an mammal in need thereof comprising administering to such mammal a macrophage activating factor prepared by contacting glycosylated mammalian vitamin D-binding protein in vitro with

.beta.-galactosidase, or

.beta.-galactosidase in combination with sialidase, .alpha.-mannosidase, or a mixture thereof.

34. A method according to claim 33 wherein the macrophage activating factor has been prepared by contacting vitamin D-binding protein in vitro with a mixture of glycosidases comprising .beta.-galactosidase, sialidase and .alpha.-mannosidase.
Description



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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
    ______________________________________


Without wishing to be bound by any theory, it is believed that the above glycosylation occurs on the protein portion of DBP through a threonine or serine residue occurring at an amino acid position corresponding to about position 420 of human DBP, or through a threonine or serine residue in the same vicinity, thus forming the O-glycosidic linkage GalNAc.alpha.(1.fwdarw.0)-Thr or GalNAc.alpha.(1.fwdarw.0)-Ser. 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 DBP and a terminal N-acetylgalactosamine group linked to an amino acid residue.

Animal DBP 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 of animal blood according to the procedure of Link et al., Anal. Biochem. 157, 262 (1986), the entire disclosure of which is incorporated herein by reference. DBP 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 DBP for actin. The entire disclosure of Haddad et al., is incorporated herein by reference. Other methods of obtaining DBP in high purity are reported in the literature The known procedures utilized for purifying the corresponding human protein, Gc protein, are directly applicable to the purification of animal DBP.

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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
    ______________________________________


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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
    ______________________________________


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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
    ______________________________________


EXAMPLE 4

A. Conversion of DBP to Macrophage Activating Factor with Immobilized Enzyme

1. Preparation of Immobilized Enzymes

100 mg of CNBr-activated agarose (Sepharose 4B) was washed with 1 mM HCl and suspended in coupling buffer (300 .mu.l) containing NaHCO.sub.3 buffer (0.1M, pH 8.3) and NaCl (0.5M). .beta.-Galactosidase, .alpha.-mannosidase or sialidase, or a combination of all three enzymes (2 U each enzyme), were mixed in 600 .mu.l of the coupling buffer and incubated at room temperature for 2 hours in an end-over-end mixer. Remaining active groups in the agarose were blocked by incubation with 0.2M glycine in coupling buffer for 2 hours at room temperature. The agarose-immobilized enzyme was washed with coupling buffer to remove unabsorbed protein and glycine, followed by washing with acetate buffer (0.1M, pH 4) containing NaCl (0.5M), and additional coupling buffer. The agarose-immobilized enzyme preparations were stored at 4.degree. C.

2. Conversion of DBP to Macrophage Activating Factor

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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
    ______________________________________


The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

* * * * *

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    ( 1 of 1 )


United States Patent 5,177,002
Yamamoto January 5, 1993

In vitro enzymatic conversion of glycosylated human vitamin D binding protein to a potent macrophage activating factor

Abstract

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.


Inventors: Yamamoto; Nobuto (1040 66th Ave., Philadelphia, PA 19126)
Assignee: Yamamoto; Nobuto (Philadelphia, PA)
Appl. No.: 576248
Filed: August 31, 1990

 
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


References Cited [Referenced By]

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U.S. Patent Documents


Other References

J. Svasti et al., Journal of Biological Chemistry 253:4188-4194, Jun. 25, 1978.
H. Van Baelen et al., Journal of Biological Chemistry 253:6344-6345, Sep. 25, 1978.
Cooke et al., J. Clin. Invest. 76:2420, 1985.
Yang et al., Proc. Natl. Acad. Sci. 82:7994, 1985.
Yamamoto et al., Cancer Res. 47:2008, 1987.
Yamamoto et al., Cancer. Immunol. Immunother. 25:185, 1987.
Yamamoto et al., Cancer Res. 24:6044, 1988.
Ngwenya et al., Abstracts of the Annual Meeting of the American Society of Microbiology, Abstract E-72, p. 121 (1988).
Homma, Abstracts of the Annual Meeting of the American Society of Microbiology, Abstract E-74, p. 121 (1988).

Primary Examiner: Wax; Robert A.
Assistant Examiner: Walsh; Stephen
Attorney, Agent or Firm: Seidel, Gonda, Lavorgna & Monaco

Parent Case Text



This is a continuation-in-part of copending application Ser. No. 439,223, filed Nov. 20, 1989 now abandoned.
Claims

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1. A process for producing a macrophage activating factor comprising contacting glycosylated human group-specific component in vitro with

.beta.-galactosidase, or

.beta.-galactosidase in combination with sialidase, .alpha.-mannosidase, or a mixture thereof,

and obtaining the macrophage activating factor.

2. A process according to claim 1 wherein the group-specific component is of phenotype Gc1, subtype Gc1f, which component is contacted with .beta.-galactosidase and sialidase.

3. A process according to claim 1 wherein the group-specific component is of phenotype Gc1, subtype Gc1s, which component is contacted with .beta.-galactosidase and .alpha.-mannosidase.

4. A process according to claim 3 wherein the group-specific component is of phenotype Gc1, variant Gc1s*, which component is contacted with .beta.-galactosidase and sialidase.

5. A process according to claim 1 wherein the group-specific component is of phenotype Gc2, which component is contacted with .beta.-galactosidase.

6. A process according to claim 1 wherein group-specific component comprising a mixture of components of types Gc1f, Gc1s and Gc2 is contacted with a mixture of glycosidases comprising .beta.-galactosidase, sialidase and .alpha.-mannosidase.

7. A process according to claim 1 wherein the enzyme or enzymes is immobilized on a solid support.

8. A process according to claim 7 wherein the solid support comprises agarose.

9. A macrophage activating factor prepared by the process of claim 1.

10. A macrophage activating factor prepared by the process of claim 2.

11. A macrophage activating factor prepared by the process of claim 3.

12. A macrophage activating factor prepared by the process of claim 4.

13. A macrophage activating factor prepared by the process of claim 5.

14. A macrophage activating factor prepared by the process of claim 6.

15. A macrophage activating factor prepared by the process of claim 7.

16. A macrophage activating composition comprising, in combination with a pharmaceutically acceptable carrier, a macrophage activating factor formed by treating glycosylated human group-specific component in vitro with

.beta.-galactosidase or

.beta.-galactosidase in combination with sialidase .alpha.-mannosidase, or mixtures thereof.

17. A macrophage activating composition according to claim 16 wherein the group-specific component is of phenotype Gc1, subtype Gc1f, which group-specific component is treated with .beta.-galactosidase and sialidase.

18. A macrophage activating composition according to claim 16 wherein the group-specific component is of phenotype Gc1, subtype Gc1s, which component is treated with .beta.-galactosidase and .alpha.-mannosidase.

19. A macrophage activating composition according to claim 16 wherein the group-specific component is of phenotype Gc1, variant Gc1s*, which component is treated with .beta.-galactosidase and sialidase.

20. A macrophage activating composition according to claim 16 wherein the group-specific component is of phenotype Gc2, which component is treated with .beta.-galactosidase.

21. A macrophage activating composition according to claim 16 wherein the group-specific component comprising a mixture of components of types Gc1f, Gc1s and Gc2 is treated with a mixture of glycosidases comprising .beta.-galactosidase, sialidase and .alpha.-mannosidase.

22. A method for inducing macrophage activation in an individual in need thereof comprising administering to such an individual macrophage activating factor prepared by contacting glycosylated human group-specific component in vitro with

.beta.-galactosidase, or

.beta.-galactosidase in combination with sialidase, .alpha.-mannosidase, or a mixture thereof.
Description



FIELD OF THE INVENTION

The invention relates to macrophage activation, in particular to the in vitro 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. 47:2008, 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). 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 stimulation of macrophages for ingestion capability (Yamamoto et al., Cancer Res. 48:6044, 1988).

B. Human Vitamin D-Binding Protein

The human vitamin D-binding protein, also known as "group-specific component" or "Gc protein", is an evolutionary conserved glycoprotein. It is a genetically polymorphic plasma protein having a relative molecular weight of about 52,000, normally constituting about 0.5% of the plasma proteins in man. The plasma concentration is generally about 260 .mu.g/ml. Polymorphism of the 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", because of a post-translational event involving sialic acid (Svasti et al., Biochem. 18:1611, 1979).

Coopenhaver et al., Arch. Biochem. Biophys. 226, 218-223 (1983) reported that the 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. While a CNBr fragment of Gc1 was found to contain N-acetylgalactosamine, no detectable galactosamine was reported in the homologous Gc2 CNBr fragment according to the method and criteria used. The Gc1 CNBr fragment further contained sialic acid, which was missing from the homologous region of Gc2.

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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
    ______________________________________


EXAMPLE 2

A. Conversion of Gc Protein to Macrophage Activating Factor with Immobilized Enzyme

1. Preparation of Immobilized Enzymes

100 mg of CNBr-activated agarose (Sepharose.RTM. 4B) was washed with 1 mM HCl and suspended in coupling buffer (300 .mu.l) containing NaHCO: buffer (0.1M, pH 8.3) and NaCl (0.5M). .beta.-Galactosidase, .alpha.-mannosidase and sialidase (2 U each enzyme) were mixed in 600 .mu.l of the coupling buffer and incubated at room temperature for 2 hours in an end-over-end mixer. Remaining active groups in the agarose were blocked by incubation with 0.2M glycine in coupling buffer for 2 hours at room temperature. The agarose-immobilized enzyme was washed with coupling buffer to remove unabsorbed protein and glycine, followed by washing with acetate buffer (0.1M, pH 4) containing NaCl (0.5M), and additional coupling buffer. The agarose-immobilized enzyme preparations were stored at 4.degree. C.

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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
    ______________________________________


The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

* * * * *

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United States Patent 5,177,004
Schutt January 5, 1993

Enzymatic deacylation of acyl-aminosorboses

Abstract

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.


Inventors: Schutt; Hermann (Wuppertal, DE)
Assignee: Bayer Aktiengesellschaft (Leverkusen, DE)
Appl. No.: 757867
Filed: September 11, 1991

 
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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References Cited [Referenced By]

U.S. Patent Documents
3880713 Apr., 1975 Fleming et al. 435/230.
3930949 Jan., 1976 Kutzbach et al. 435/230.
3962036 Jun., 1976 Liersch et al. 435/230.
4264734 Apr., 1981 Kahan et al. 435/228.
4282322 Aug., 1981 Kahan et al. 435/228.
4405714 Sep., 1983 Kinast et al. 435/84.
4806650 Feb., 1989 Schroder et al. 435/84.
4981789 Jan., 1991 Lein 435/228.
Foreign Patent Documents
0049858 Apr., 1982 EP.
0240868 Oct., 1987 EP.

Primary Examiner: Lilling; Herbert J.
Attorney, Agent or Firm: Sprung Horn Kramer & Woods

Claims



1. A process for preparing aminosorbose comprising enzymatically deacylating N-phenylacetyl-aminosorbose with pencillin acylase at a pH of from about 8 to 9.5.
Description



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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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United States Patent 5,326,749
Yamamoto * July 5, 1994

Macrophage activating factor from vitamin D binding protein

Abstract

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.


Inventors: Yamamoto; Nobuto (1040 66th Ave., Philadelphia, PA 19126)
[*] Notice: The portion of the term of this patent subsequent to August 31, 2010 has been disclaimed.
Appl. No.: 000320
Filed: January 4, 1993

 
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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References Cited [Referenced By]

U.S. Patent Documents
5177001 Jan., 1993 Yamamoto 435/68.
5177002 Jan., 1993 Yamamoto 435/68.


Other References

Yamamoto et al., Cancer Research 47:2008, 1987.
Yamamoto et al., Cancer Immunol. Immunother. 25:185, 1987.
Yamamoto et al., Cancer Res. 48:6044, 1988.
Ngwenya et al., Abstracts of the Annual Meeting of the American Society of Microbiology, Abs. E-72, p. 121 (1988).
Homma, Abstracts of the Annual Meeting of the Am. Society of Microbiology, Abs. E-74, p. 121 (1988).
Cooke et al., J. Clin. Invest. 76:2420-2424 (1985).
Yang et al., Proc. Natl. Acad. Sci. 82:7994-7998 (1985).
Biological Abstracts 68(5) p. 2831 (1979) Abs. No. 28360.
Yamamoto, et al., Proc. Natl. Acad. Sci. USA 88, 8539-8543 (1991).
Haddad et al., Biochem. J. 218:805, 1984.
Link et al., Analyt. Biochem. 157: 262-269, 1986.
Cooke et al., Endocrine Reviews 10:294-307, 1989.
Ogata et al., Comp. Biochem. Physiol. 90B:193-199, 1988.
Van De Weghe et al., Comp. Biochem. Physiol. 73B977-982, 1982.
Van Baelen et al. J. Biol. Chem. 253:6344-6345, Sep. 25, 1978.
Svasti et al., J. Biol. Chem. 253:4188-4194, Jun. 25, 1978.
Shinomiya et al., J. Biochem. 92:1163-1171, 1982.
Gahne et al., Anim. Blood Groups Biochem. Genet. 9, 37-40 (1978).
Homma and Yamamoto, Clin. Exp. Immunol. 79, 307-313 (1990).
Homma et al., Immunol. Cell. Biol. 68, 137-142 (1990).
Yamamoto et al., Immunology 74, 420-424 (1991).
Yamamoto et al., J. Immunol. 147, 273-280 (Jul. 1, 1991).
Viau et al., "Isolation and characterization of the O-glycan chain of the human vitamin-D binding protein", Bch. B Phys. Res. Comm., 117:324-331 (1983).
Coppenhaver et al., "Post-Translational Heterogeneity of the Human Vitamin D-Binding Protein", Arch. Biochem. Biophys. 226(1):218-223 (1983).
Schulz et al., Principles of Protein Structure, pp. 14-16 (1979).

Primary Examiner: Wax; Robert A.
Assistant Examiner: Walsh; Stephen G.
Attorney, Agent or Firm: Seidel, Gonda, Lavorgna & Monaco

Parent Case Text



This application is a continuation-in-part of application 07/767,742, filed Sep. 30, 1991, now U.S. Pat. No. 5,177,001, and a continuation in part of application 07/576,248, filed Aug. 31, 1990, now U.S. Pat. No. 5,177,002, which is a continuation in part of application 07/439,223, filed Nov. 20, 1989, abandoned.

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Claims



1. A macrophage activating factor comprising an isolated and purified polypeptide having the amino acid sequence of vitamin D-binding protein, and a terminal N-acetylgalactosamine group linked to a threonine or serine residue of said polypeptide at amino acid position 418.

2. A macrophage activating factor comprising an isolated and purified polypeptide having the amino acid sequence of vitamin D-binding protein, and a terminal N-actylgalactosamine group linked to a threonine residue of said polypeptide at amino acid position 420.

3. A pharmaceutical composition comprising a macrophage activating effective amount of the macrophage activating factor of claim 1 and a pharmaceutically acceptable carrier.

4. A pharmaceutical composition comprising a macrophage activating effective amount of the macrophage activating factor of claim 2 and a pharmaceutically acceptable carrier.
Description



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. 47:2008, 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 macrophages 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 (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). 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).

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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).

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 actinagarose 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 vitamin D-binding protein, and the existence of ready methods for its purification, the 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. Vitamin D-binding protein, which is an evolutionary conserved protein, is a precursor of the macrophage activating factor. 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, 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, DBP, which possesses 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 DBP with all three enzymes to obtain the macrophage activating factor, particularly when DBP purified from pooled plasma of different individuals is utilized.

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The invention also relates to a macrophage activating factor which may be 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 pharmaceutical or veterinary use.

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

"DBP" as used herein means the genetically polymorphic vitamin D-binding protein also known as "group specific component" ("Gc") in humans, 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.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B contain the reported amino acid sequence of DBPgs/gm. The underlined amino acid residues at positions 152, 311, 416 and 420 differ from DBPg.

FIGS. 2A and 2B contain the reported amino acid sequence of DBPg. The underlined amino acid residues at positions 152, 311, 416 and 420 differ from DBPgs/gm.

DETAILED DESCRIPTION OF THE INVENTION

A serum factor, which has been identified as 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 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 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 mammalian 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.

The molecules of the DBPgs and DBPgm penotypes on the one hand (Gc1 in humans) is believed to differ from the DBPg phenotype (Gc2 in humans) by four amino acids at positions 152, 311, 416 and 420, as reported in the literature and reproduced in FIGS. 1 and 2. The differences are as follows:

    ______________________________________
                   152   311     416     420
    ______________________________________
    DBPgs, DBPgm (Gc1)
                     Glu     Arg     Glu   Thr
    DBPg (Gc2)       Gly     Glu     Asp   Lys
    ______________________________________


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All three principal DBP types--DBPgm, DBPGs and DBPg--differ in the nature of the appended oligosaccharide, although it is believed that most DBP molecules having the amino acid sequence of DBPg molecules are unglycosylated. Only the glycosylated form of DBPg is a precursor for macrophage activating factor according to the process described herein. Incubation of each of DBPgm, DBPgs and DBPg 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 galactose.

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 individuals. 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.

The innermost sugar of the oligosaccharide moiety of DBPgs and DBPgm protein is N-acetylgalactosamine (Coppenhaver et al., Arch. Biochem. Biophys. 226, 218-223, 1983). Treatment of these glycoproteins 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.

It is believed that 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 Species
    ______________________________________
    DBPgs
             ##STR1##       human (Gc1f, Gc1s*), monkey, bovine, sheep, goat,
                            pig, horse
    DBPgm
             ##STR2##       human (Gc1s, Gc1f*), bovine
    DBPg    GalGalNAc       human (Gc2), dog, cat,
                            rat, mouse
    ______________________________________


It is believed that the above glycosylation occurs on the protein portion of DBP through a threonine or serine residue occurring at an amino acid position corresponding to about position 420 of human DBP, or through a threonine or serine residue in the same vicinity, thus forming the O-glycosidic linkage GalNAc.alpha.(1.fwdarw.0)Thr or GalNAc.alpha.(1.fwdarw.0)-Ser. Thus, the novel macrophage activating factor comprises a protein in substantially pure form having substantially the amino acid sequence of DBP and a terminal N-acetylgalactosamine group linked to an amino acid residue in the vicinity of amino acid 420, such as Thr(418) in human DBPg (Gc2); or Thr(418) (or Thr(420)) in DBPgs/gm; or Ser(418) in those species of DBg, such as rat and mouse DBg, which contain serine at position 418 in lieu of threonine.

DBP 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 of animal blood according to the procedure of Link et al., Anal. Biochem. 157, 262 (1986), the entire disclosure of which is incorporated herein by reference. DBP 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 DBP for actin. The entire disclosure of Haddad et al., is incorporated herein by reference. Other methods of obtaining DBP in high purity are reported in the literature.

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.

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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.

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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 mammalian 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 mammalian species would receive only macrophage activating factor derived from the blood of the same species. Similarly, the risk of immunologic in individuals 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, a mammal 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.

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EXAMPLE 1

A. Conversion of Human DBP (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.

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

Gcls 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 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.

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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EXAMPLE 2

Conversion of Human DB (Gc Protein) to Macrophage Activating Factor with Immobilized Enzyme

1. Preparation of Immobilized Enzymes

100 mg of CNBr-activated agarose (Sepharose.RTM. 4 B) was washed with 1 mM HCl and suspended in coupling buffer (300 .mu.l) containing NaHCO.sub.3 buffer (0.1M, pH 8.3) and NaCl (0.5M). .beta.-Galactosidase, .alpha.-mannosidase and sialidase (2 U each enzyme) were mixed in 600 .mu.l of the coupling buffer and incubated at room temperature for 2 hours in an end-over-end mixer. Remaining active groups in the agarose were blocked by incubation with 0.2M glycine in coupling buffer for 2 hours at room temperature. The agarose-immobilized enzyme was washed with coupling buffer to remove unabsorbed protein and glycine, followed by washing with acetate buffer (0.1M, pH 4) containing NaCl (0.5M), and additional coupling buffer. The agarose-immobilized enzyme preparations were stored at 4.degree. C.

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
    enzymatically
    modified Ingestion Index
    Gc protein
             Untreated       Glycosidase-
    (picogram/
             Control         treated
    mouse)   Gc1      Gc1 + Gc2  Gc1    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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EXAMPLES 3

A. Conversion of Animal 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 MgSO.sub.4 and treated with 2 .mu.l of PBS-Mg containing 0.1 U of the enzyme combinations indicated in Table 4. 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.

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 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 4.

                  TABLE 4
    ______________________________________
    Macrophage Activation by
    Glycosidase-treated DBP
             Ingestion Index
               A         B         C      D
    Glycosidases for     pooled
    treatment of DBP
               bovine    bovine    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
    .alpha.-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
    .alpha.-mannosidase
    ______________________________________


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It is apparent from Table 4 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 4 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 3, 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 5 (bovine DBPgs), Table 6 (pooled bovine DBP) and Table 7 (cat DBPg).

                  TABLE 5
    ______________________________________
    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 6
    ______________________________________
    Macrophage Activation by
    Glycosidase-treated pooled bovine DBP
            Ingestion Index
    Dilution of                      Bovine DBP
    Glycosidase-         Bovine DBP  treated with
    Treated pooled
              Bovine DBP treated with
                                     .beta.-galactosidase
    Bovine DBPgs
              untreated  .beta.-galactosidase
                                     and .alpha.-
    and DBPgm control    and sialidase
                                     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 7
    ______________________________________
    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
    ______________________________________


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EXAMPLE 4

Purified DBP (1.0 .mu.g from each of the species identified in Table 8, below, was treated according to Example 3 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 3B. The results are set forth in Table 8. 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 8
    ______________________________________
                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
    ______________________________________


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EXAMPLE 6

A. Conversion of DBP to Macrophage Activating Factor with Immobilized Enzyme

1. Preparation of Immobilized Enzymes

100 mg of CNBr-activated agarose (Sepharose 4B) was washed with 1mM HCl and suspended in coupling buffer (300 .mu.l) containing NaHCO.sub.3 buffer (0.1M, pH 8.3) and NaCl (0.5M). .beta.-Galactosidase, .alpha.-Mannosidase or sialidase, or a combination of all three enzymes (2 U each enzyme), were mixed in 600 .mu.l of the coupling buffer and incubated at room temperature for 2 hours in an end-over-end mixer. Remaining active groups in the agarose were blocked by incubation with 0.2M glycine in coupling buffer for 2 hours at room temperature. The agarose-immobilized enzyme was washed with coupling buffer to remove unabsorbed protein and glycine, followed by washing with acetate buffer (0.1M, pH 4) containing NaCl (0.5M), and additional coupling buffer. The agarose-immobilized enzyme preparations were stored at 4.degree. C.

2 Conversion of DBP to Macrophage Activating Factor

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 3B.2. were layered onto the coverglass, and a phagocytosis assay was performed as in Example 3B.3. The results are set forth in Table 9:

                  TABLE 9
    ______________________________________
    In Vivo Assay of Macrophage Activation
    by Glycosidase-treated Bovine DBPgs
           Ingestion Index
    Dosage of
             Bovine DBP      Dog DBP
    enzymatically     treated with      treated with
    modified          .beta.-galacto-   .beta.-galacto-
    DBP (pico-
             untreated
                      sidase and untreated
                                        sidase and
    gram/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
    ______________________________________

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

* * * * *

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    ( 1 of 1 )

United States Patent 5,837,252
Sinnott ,   et al. November 17, 1998

Nontoxic extract of Larrea tridentata and method of making same

Abstract

A nontoxic, therapeutic agent having pharmacological activity comprising concentrated extract of Larrea tridentata plant material and ascorbic acid, an ascorbic acid ester, an ascorbic acid salt, butylated hydroxyanisole, butylated hydroxytoluene, hydrogen sulfide, hypophosphorous acid, monothioglycerol, potassium bisulfite, propyl gallate, sodium bisulfite, sodium hydrosulfite, sodium thiosulfate, sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E is made by a process in which the plant material is extracted using an organic solvent, preferably acetone, and is then saturated with one of the listed reducing agents acid to reduce the toxic NDGA quinone, which naturally occurs in the plant material, to NDGA itself. Additional amounts of ascorbic acid, an ascorbic acid ester, an ascorbic acid salt, butylated hydroxyanisole, butylated hydroxytoluene, hydrogen sulfide, hypophosphorous acid, monothioglycerol, potassium bisulfite, propyl gallate, sodium bisulfite, sodium hydrosulfite, sodium thiosulfate, sulfur dioxide, sulfurous acid, a tocopherol, or vitamn E may be added to the extract to inhibit the natural oxidation of the NDGA into the toxic NDGA quinone in vivo, or during processing or storage. The resulting extract is useful in the treatment of viral diseases caused by viruses from the Herpesviridae family or viruses which require the Sp1 class of proteins to initiate viral replications. The resulting compound can also be used as an anti-inflammatory when the inflaatory diseases are mediated by the effects of leukotrienes. The listed reducing agents can also be used to stabilize NDGA as a therapeutic agent or a food additive.

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Inventors: Sinnott; Robert A. (Chandler, AZ); Clark; W. Dennis (Phoenix, AZ); DeBoer; Kenneth Frank (Belgrade, MT)
Assignee: Larreacorp, Ltd. (Chandler, AZ)
Appl. No.: 726686
Filed: October 7, 1996

 
U.S. Class: 424/195.1
Intern'l Class: A01N 065/00
Field of Search: 424/195.1


References Cited [Referenced By]

U.S. Patent Documents
2373192 Apr., 1945 Lauer 426/546.
2382475 Aug., 1945 Gisvold 568/729.
2644822 Jul., 1953 Pearl 549/435.
4765927 Aug., 1988 Nomura et al. 252/400.
4774229 Sep., 1988 Jordan 514/25.
4880637 Nov., 1989 Jordan 424/641.
5276060 Jan., 1994 Neiss et al. 514/731.


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Other References

Timmermann "Practical uses of Larrea,' in Creosote Bush: Biology and Chemistry of Larrea in the New World Deserts," Mabry et al. eds. (1977) (Dowden, Hutchison and Ross: Pennsylvania) pp. 252-276.
Tierra, L., "Chapparal," The Herbs of Life: Health and Healing Using Western and Chinese Techniques, pp. 561, 180-181 (1992).
Crellin, J.K. et al., "Chapparal," Herbal Medicines Past and Present II: A Reference Guide to Medicinal Plants, pp. 150-151 (1990).
Sakakibara, et al., Flavonoid Methyl Esters on the External Leaf Surface of Larrea Tridentata and L. Divaricata, 1976, pp. 727-731.
Obermeyer, et al., Chemical Studies of Phytoestrogens and Related Compounds in Dietary Supplements: Flax and Chaparral, 1995, pp. 6-10 vol. 208.
Phytochemistry vol. 23(6) Donald MacRae, et al., Biological Activities of Lignans, 1984, pp. 1207-1220.
Proc. Natl. Acad. Sci. USA vol. 92, Gnabre et al., Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans, 1995, pp. 11239-11243.
Gnabre et al., Characterization of Anti-HIV Lignans from Larrea tridentata, 1995, pp. 12203-12210, Tetrahedron vol. 51(4).
Gnabre et al., Isolation of anti-HIV-1 lignans from Larrea tridentata by counter-current chromatography, 1996, pp. 353-364 J. Chromatography vol. 719.
Amer. Soc. Hematol. vol. 88(2) Nador et al., Primary Effusion Lymphoma: A Distinct Clinicopathologic Entity Associated With the Kaposi's Sarcoma--Associated Herpes Virus, 1996, pp. 645-656.
The Philadelphia Inquirer Crenson, Kaposi's Sarcoma is tied to herpes, Jul. 31, 1996, p. A4.
Altman, Aids Cancer Said to Have Viral Source: Breakthrough Seen in Kaposi's Sarcoma, Feb. 1, 1995, p. A22 The New York Times.
Critchfield et al., Inhibition of HIV Activation in Latently Infected Cells by Flavonoid Compounds in AIDS Research and Human Retroviruses, 1996, pp. 39-46 vol. 12(1).
Amer. College Phys. vol. 121 Henderson, The Role of Leukotrienes in Inflammation, 1994, pp. 684-697.
Israel et al., Effect of Treatment With Zileuton, a 5-Lipoxygenase Inhibitor, in Patients With Asthma, 196, pp. 932-936 JAMA vol. 275(12).
Gordon, The Microbicical Potential of Various Creosotebush (Larrea Tridentata) Extracts, 1987, pp. 83-85 vol. 21, J. Arizona--Nevada Acad. Sci.

Primary Examiner: Witz; Jean C.
Assistant Examiner: Hanley; Susan
Attorney, Agent or Firm: Benson; David K., Nichols; Steven L.

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Claims



1. A method of preparing a medicinally effective, nontoxic composition containing an extract of Larrea tridentata plant material produced by the steps of:

harvesting pant material comprising leaves from Larrea tridentata shrubs;

air drying said harvested plant material at ambient temperature and humidity for at least one week;

maintaining said plant material in whole form without chopping, grinding or powdering;

extracting said Larrea tridentata plant material in whole form by recirculating acetone over said plant material at least three times to produce a Larrea tridentata extract which comprises NDGA quinone;

filtering particulate impurities from said extract;

adding polysorbate 80 to said extract as an emulsifying and stabilizing agent, wherein 5 mL of polysorbate 80 are added per 50 L of extract;

reducing said NDGA quinone in said extract to NDGA by passing said extract through a column packed with powdered ascorbic acid to produce a reduced extract, wherein 5 g of ascorbic acid powder are provided per liter of extract being reduced;

concentrating said reduced extract by boiling off the acetone solvent at about 100.degree.C. until the volume of said extract is reduced by 90% to produce a concentrated extract;

adding additional amounts of ascorbic acid effective to prevent oxidation of said NDGA in said concentrated extract; and

optionally, combining said concentrated extract with a pharmaceutically acceptable carriers, excipients and/or agents.

2. The method of claim 1, wherein said concentrated extract is combined with a pharmaceutically acceptable carrier to make a lotion for topical treatment of herpes lesions.

3. The method of claim 1, wherein said concentrated extract is combined with an extract of Echinacea or Podophyllin.

4. The method of claim 1, wherein said concentrated extract is combined with a dry excipient material selected from the group consisting of starch, sucrose and fructose to make a pharmaceutical composition.

5. The method of claim 4, wherein said pharmaceutical composition is encapsulated.

6. The method of claim 1, wherein said concentrated extract is combined with an anti-viral or anti-inflammatory agent.

7. A method of treating herpes lesions comprising topically applying to the affected area of a human in need thereof, an effective amount of an extract of Larrea tridentata.

8. A method of treating herpes virus comprising administering to a human in need thereof, an effective amount of a pharmaceutical composition of an extract of Larrea tridentata.
Description



This application claims the benefit of U.S. Provisional application Ser. No. 60/946, filed Jul. 1, 1996.

FIELD OF THE INVENTION

The present invention relates generally to a nontoxic extract of Larrea tridentata plant material having therapeutic value and a method of making the same. The present invention also relates to the field of nontoxic, stable NDGA products used as food additives and therapeutic agents.

BACKGROUND OF THE INVENTION

Larrea tridentata, also known as Larrea divaricata, Larrea, chaparral, or creosote bush, is a shrubby plant which dominates some areas of the desert southwest in the United States and Northern Mexico as well as some desert areas of Argentina. Tea made from the leaves of Larrea tridentata has long been used in folk medicine to treat digestive disorders, rheumatism, venereal disease, sores, bronchitis, chicken pox, and the common cold.

According to Masayuki Sakakibara, et al., Flavonoid Methyl Ethers on the External Leaf Surface of Larrea Tridentata and L. Divaricata in PHYTOCHEMISTRY, vol. 15, pp. 727-731 (Pergamon Press 1976), the disclosure of which is incorporated herein by reference, the natural products on the surface of the Larrea tridentata leaves, the leaf resin, constitutes approximately 10-15% of the dry weight of the leaves and is composed of approximately 50% nordihydroguaiaretic acid ("NDGA") and related lignans, and 50% flavonoids. NDGA, extracted from Larrea tridentata by an alkaline extraction method (U.S. Pat. No. 2,382,475, incorporated herein by reference) and produced synthetically (U.S. Pat. No. 2,644,822, incorporated herein by reference) was used as an antioxidant in edible fats, butter, oils and oleaginous materials (U.S. Pat. No. 2,373,192, incorporated herein by reference), until the GRAS (Generally Recognized As Safe) status of NDGA was revoked after animal studies revealed evidence of kidney toxicity resulting from the ingestion of NDGA.

NDGA is known as a powerful antioxidant compound. However, NDGA can itself be oxidized to toxic oxidation products by chemical means or by oxidation during processing and storage. A highly reactive and toxic oxidation product of NDGA is nordihydroguaiaretic acid ortho di-a-b-unsaturated quinone ("NDGA quinone"), which according to T. J. Mabry et al.,The Natural Products Chemistry of Larrea in CREOSOTE BUSH: BIOLOGY AND CHEMISTRY OF LARREA IN THE NEW WORLD DESERTS, ch. 5, pp. 115-133 (Dowden, Hutchinson and Ross, Pennsylvania 1977) (incorporated herein by reference), occurs in Larrea and Larrea extracts and probably serves as a toxin to protect the plant from being eaten by herbivores. According to a recent report by FDA scientists (W. R. Obermeyer et al., Chemical Studies of Phytoestrogens and Related Compounds in Dietary Supplements: Flax and Chaparral, 208 PROC. SOC. EXP. BIOL. MED., pp. 6-12 (1995), the disclosure of which is incorporated herein by reference), NDGA quinone is found in chaparral (Larrea tridentata) and is suspected to be a causative agent of the toxic effects associated with consumption of chaparral products.

NDGA is the dominant lignan present in Larrea tridentata. NDGA is known to possess a variety of biological effects including anti-tumor activity, enzyme inhibition activity and antimicrobial activity according to W. Donald MacRae & G. H. Neil Towers, Biological Activities of Lignans , PHYTOCHEMISTRY, vol. 23, pp. 1207-1220 (Pergamon Press 1984) (incorporated herein by reference). Additionally, NDGA and other antioxidants have been shown to be potent inhibitors of the human immunodeficiency virus type 1 (HIV) transcription The mode of action of this anti-HIV activity was suggested to be due to the potent antioxidant activity of NDGA inhibiting a redox regulated signal transduction pathway leading to production of HIV virus.

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More recently, three scientific articles, John N. Gnabre et al., Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans, PROC. NATL. ACAD. SCI., USA, vol 92, pp.11239-11243 (November 1995); John Gnabre et al., Characterization of Anti-HIV Lignans from Larrea tridentata, TETRAHEDRON, vol. 51, pp. 12203-12210 (1995); and John Noel Gnabre et al., Isolation of anti-HIV lignans from Larrea tridentata by counter-current chromatography, JOURNAL OF CHROMATOGRAPHY A, vol. 719, pp. 353-364 (1996), the disclosure of all three articles being incorporated herein by reference, demonstrate that at least two lignans isolated from Larrea tridentata, NDGA and 3-O-methyl nordihydroguaiaretic acid ("Mal. 4") inhibit transcription and replication of human immunodeficiency virus type 1 by a novel mechanism. This elucidated mechanism of anti-HIV activity is thought to be due to the ability of the two identified Larrea tridentata lignans, NDGA and Mal. 4, to interfere with the binding of the Sp1 protein to Sp1 binding sites in the HIV long terminal repeat (HIV-LTR). According to this theory, by inhibiting Sp1 binding in the HIV-LTR, the promoter activity of the HIV-LTR is eliminated so that HIV transcription, HIV Tat-regulated transactivation and HIV replication do not occur. It is further theorized by the authors that viruses other than HIV, which require binding of Sp1 protein in promoter-containing Sp1 binding sites to initiate viral replication, might also be inhibited by the anti-HIV lignans isolated from Larrea tridentata and that this class of lignans, in general, may possess a broader antiviral action of important interest.

Also, in a recent article, Anneke K.Raney & Alan McLachlan, Characterization of the Hepatitis B Virus Large Surface Antigen Promoter Sp1 Binding Site, VIROLOGY, vol. 208, pp. 399-404 (1995), binding sites for the transcription factor Sp1 have been identified in the DNA promoter regions of at least two important viral genes of the Hepatitis B virus (HBV) which may be involved in the coordinate regulation of HBV transcription by transcription factor Sp1.

Kaposi's Sarcoma, a cancer that frequently occurs among AIDS patients, has recently been implicated to be caused by a new herpes virus, human herpes virus-8 (HHV-8). See Roland G. Nador et al., Primary Effusion Lymphoma: A Distinct Clinicopathologic Entity Associated With the Kaposi's Sarcoma--Associated Herpes Virus, BLOOD, vol. 88, no. 2, pp. 645-656 (Jul. 15, 1996), incorporated herein by reference. See also, Matt Crenson, Kaposi's Sarcoma is tied to herpes, THE PHILADELPHIA INQUIRER, p. A4 (Jul. 31, 1996), and Lawrence K. Altman, Aids Cancer Said to Have Viral Source: Breakthrough Seen in Kaposi's Sarcoma, NEW YORK TIMES .sctn. A, p. 22 (Feb. 1, 1995), incorporated herein by reference.

Certain flavonoid compounds, especially members of the chemical classes flavones and flavonols can inhibit HIV activation at fairly low concentrations (See , J. William Critchfield et al, Inhibition of HIV Activation in Latently Infected Cells by Flavonoid Compounds in AIDS Research and Human Retroviruses, AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 12, no. 1, pp. 39-46 (1996), incorporated herein by reference). As further cited in Masayuki Sakakibara, et al., Flavonoid Methyl Ethers on the External Leaf Surface of Larrea Tridentata and L. Divaricata in PHYTOCHEMISTRY, vol. 15, pp. 727-731 (Pergamon Press 1976), Larrea tridentata contains an abundance of these classes of antiviral flavonoids, particularly methyl ethers of flavonols.

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Like many physiologically active chemicals isolated from plant sources, these antiviral lignans and flavonoid compounds appear to work synergistically with other unresolved compounds present in crude extracts of Larrea tridentata. The identity and mode of action of these synergistic compounds is unknown but they may facilitate absorption of the antiviral lignans or otherwise enhance the specific physiological, antiviral effects.

NDGA is known to be a potent inhibitor of the enzyme 5-lipoxygenase. One of the enzymatic products of 5-lipoxygenase is 5-hydroperoxyeicosatetraenoic acid (HPETE) which is the precursor compound for the biosynthesis of very potent chemical mediators of inflammation, known as leukotrienes. As detailed in William R. Henderson, The Role of Leukotrienes in Inflammation, ANN. INTER. MED., vol. 121, pp. 684-697 (1994), the disclosure of which is incorporated herein by reference, 5-lipoxygenase is limited to a specific number of myeloid cells including: neutrophils, eosinophils, monocytes, macrophages, mast cells, basophils and B-lymphocytes. Leukotrienes are chemicals which induce prolonged muscle contraction, especially in the bronchioles of the lungs, and also increase vascular permeability and attract neutrophils and eosinophils to the site of inflammation. The leukotrienes play a major role in the inflammatory response to injury. Leukotrienes have also been implicated in the pathogenesis of several inflamatory diseases including: asthma, psoriasis, rheumatoid arthritis and inflammatory bowel disease. The role of leukotrienes as mediators of inflammatory diseases makes them attractive targets for therapeutic drugs to treat these diseases.

Many inhibitors of leukotriene synthesis are being developed. Recently, a 5-lipoxygenase inhibitor, Zileuton, was found to be effective in the treatment of asthma during clinical tests (Elliot Israel et al., Effect of Treatment With Zileuton, a 5-Lipoxygenase Inhibitor, in Patients With Asthma, JAMA, vol. 275, pp. 931-936 (Mar. 27, 1996), the disclosure of which is incorporated herein by reference). The success of Zileuton underscores the utility and need of therapeutic agents containing 5-lipoxygenase inhibitors in the treatment of inflammatory disease processes, including asthma

Throughout this specification and claims, viral diseases are intended to include all diseases, attributed to a pathological virus of humans or animals, in which the causative viral agent which requires the Sp1 class of proteins to initiate viral replication, including certain viral agents of venereal diseases such as the Herpes viruses (the Herpesviridae), HSV-1 and HSV-2, viral hepatitis (the Hepadnaviridae) such as hepatitis B, and members of the retrovirus family (the retroviridae) including Varicella-Zoster viruses, cytomegalovirus (CMV), the human T-lymphotrophic viruses 1 and 2 (HTLV-1 and (HTLV-2) the human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2) and the cancer Kaposi's Sarcoma. Inflammatory diseases, throughout the specification and claims, are intended to include all diseases in which leukotrienes are known to play a major role or have been implicated including: asthma, allergic rhinitis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, inflammatory pain, cystic fibrosis, adult respiratory distress syndrome, glomerulonephritis, inflammation of the skin, and virally induced inflammation (caused by CMV and other members of the Herpesviridae) leading to atherosclerosis/ arteriosclerosis and subsequent coronary artery disease.

In light of the foregoing background, there exists the need for a commercial method of producing a Larrea tridentata extract which contains a high concentration of both the identified antiviral lignans (NDGA and Mal. 4), flavonoids, and a wide variety of other associated organic compounds from the leaf resin, which may contribute synergistic antiviral and lipoxygenase inhibitory activity.

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Additionally, for the purpose of toxicological safety, it is of critical importance that the Larrea tridentata extract, to be used for medical applications, be processed to reduce the concentration of the toxic compound, NDGA quinone, which is reported to occur naturally in Larrea tridentata plant tissues. There is also a need to inhibit the natural production of toxic oxidation products, such as NDGA quinone, in the Larrea tridentata extract during processing and storage of the extract and formulated products and to facilitate the processing of the concentrated extract as either a liquid, slurry, or solid.

Lastly, there is a need for products comprising synthesized NDGA which is stabilized against oxidation into NDGA quinone during processing and storage.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to meet the above described needs and others. It is an object of the present invention to provide a nontoxic extract of Larrea tridentata having a high concentration of NDGA. It is a further object of the present invention to provide a nontoxic extract of Larrea tridentata containing Mal.4 and other compounds having known or expected, and perhaps synergistic, therapeutic effects.

It is a further object of the present invention to provide such an extract which contains little or no NDGA quinone and which inhibits the production of NDGA quinone in vivo and during processing and storage. It is a further object of the present invention to provide a method of making the above-described extract, and formulations thereof. It is a further object of the present invention to describe some of the potential therapeutic uses of the above-described extract, and formulations thereof.

It is a further object of the present invention to provide NDGA products for use as food additives or therapeutic agents in which the NDGA is prevented during storage and processing from oxidizing into NDGA quinone.

Additional objects, advantages and novel features of the invention will be set forth in the description which follows or may be learned by those skilled in the art through reading these materials or practicing the invention. The objects and advantages of the invention may be achieved through the means recited in the attached claims.

To achieve the stated and other objects of the present invention, as embodied and described below, the invention may comprise:

A method of preparing a nontoxic extract of Larrea tridentata plant material comprising the steps of:

extracting endogenous antiviral and anti-inflammatory lignans and flavanoids and other synergistic compounds from Larrea tridentata plant material with a polar solvent, preferably acetone, to produce an extract by recirculating the solvent over the plant material a plurality of times;

filtering the extract;

adding an emulsifying and stabilizing agent, preferably polysorbate 80, to the filtered extract;

reducing the NDGA quinone in the extract with ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 mercapto-1,2-propanediol), potassium bisulfite potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol), preferably ascorbic acid, by passing the extract through a bed of ascorbic acid powder;

boiling the organic solvent out of the compound; and

adding additional amounts of one of the above listed reducing agents to the compound subsequent to the step of reducing.

The present invention may also comprise:

A nontoxic, therapeutic agent comprising:

an extract of Larrea tridentata plant material comprising NDGA and Mal.4; and

ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 mercapto-1,2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol).

The present invention may also comprise:

formulations of the described therapeutic agent in the embodiment of a lotion, liquid, powder or pill.

The present invention may also comprise a food additive or therapeutic agent comprising:

NDGA; and

ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 mercapto-1,2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol)

The present invention may also comprise:

A method of treating:

Viral diseases caused by viruses from the retrovirus family of viruses, the Hepatitis B virus, the Herpesviridae family of viruses, and viruses which require Sp1 class proteins to initiate viral replication,

inflammatory diseases which are mediated by the effects of leukotrienes, and virally induced inflammation leading to atherosclerosis, hypertension, atherosclerosis, arteriosclerosis, and subsequent coronary artery disease

using a nontoxic, therapeutic agent comprising:

an extract of Larrea tridentata plant material comprising NDGA and Mal. 4; and

ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3-mercapto-1,2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol).

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BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate the present invention and are a part of the specification. Together will the following description, the drawings demonstrate and explain the principles of the present invention. In the drawings:

FIG. 1 is a high performance liquid chromatography ("HPLC") tracing of NDGA, as a purified compound (reported by the manufacturer to have a minimum purity of 90%). Slight amounts of unidentified impurities, probably methylated NDGA compounds, are indicated. However, no NDGA quinone is detectable in the sample. The tracing was taken at 280 nm absorbance.

FIG. 2 is a HPLC tracing of NDGA as a purified compounds described in reference to FIG. 1 which has been treated with the strong oxidizing agents sulfuric acid and potassium dichromate. NDGA quinone, the oxidation product of NDGA, is identified in the chromatogram as the large peak at 16.5 minutes. The tracing was taken at 280 nm absorbance.

FIG. 3 is an HPLC tracing of a purified NDGA sample as described in reference to FIG. 1 and treated with oxidizing agents as described in reference to FIG. 2, and then treated according to the principles of the present invention, with the chemical reducing agent, ascorbic acid. NDGA quinone is not detectable at 16.5 minutes after treatment was completed. The tracing was taken at 280 nm absorbance.

FIG. 4 is an HPLC tracing of a concentrated Larrea tridentata extract, which has been treated with the strong oxidizing agents sulfuric acids and potassium dichromate. A peak corresponding with NDGA quinone is identified in the chromatogram as the small peak occurring at 30 minutes. The tracing was taken at 375 nm absorbance.

FIG. 5 is an HPLC tracing of a concentrated Larrea tridentata extract, produced according to the methods of the present invention. No NDGA quinone peak is observed at 30 minutes. The tracing was taken at 375 nm absorbance.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention includes at least two products.

(1) a nontoxic extract of Larrea tridentata plant material having a high concentration of NDGA and little or no NDGA quinone which can be used as a therapeutic agent; and

(2) NDGA, for use as a food additive or therapeutic agent, which does not oxidize into NDGA quinone during storage or processing.

An important principle of the invention is that ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 mercapto-1,2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol), when combined with NDGA, reduce any NDGA quinone present into NDGA, and prevent the NDGA from oxidizing and producing more NDGA quinone. These agents are not necessarily considered to be equivalents to each other, some having advantages not possessed by all the others. It is only asserted that each of these listed agents may be used according to the principles of the invention as described in this specification to substantially accomplish the objects of the invention. The preferred embodiment of the invention will be described below.

According to the principles of the present invention, a nontoxic extract of Larrea tridentata having a high concentration of NDGA and very little or no NDGA quinone can be prepared by saturating the extract with one or more of the above listed agents. Additional amounts of one of the listed agents may be added to the extract or products formulated therefrom to inhibit the natural oxidation of component NDGA into NDGA quinone during processing and storage.

According to the preferred embodiment of the present invention, a nontoxic extract is prepared by the following method. Larrea tridentata plant material consisting mostly of leaves and stems, but also possibly containing a small amount of flowers and fruits, i.e. whole plant material, is air dried. The dried plant material is extracted using a suitable solvent. In the preferred embodiment, the solvent is an organic solvent, preferably acetone.

The organic solvent is recirculated three times over the correct ratio of plant material to completely dissolve the organic compounds on the surface of the plant material and to produce a crude Larrea tridentata extract. The resulting crude extract is filtered through cellulosic media (i.e. qualitative grade filter paper) to remove dirt and particulates. A chemical emulsifying and stabilizing agent, Food Chemicals Codex (F.C.C.) grade polysorbate 80, is added to the clarified extract at a concentration of 0.01% by volume.

The clarified extract is then passed through a bed of ascorbic acid powder (5 grams of F.C.C. grade ascorbic acid powder for each liter of extract passed through) in a manner which facilitates contact of the extract with the ascorbic acid powder and results in saturation of the extract with ascorbic acid. Saturating the extract with the chemical reducing agent, ascorbic acid results in conditions which favor the chemical reduction of the toxic, oxidative metabolites of NDGA, which are present in Larrea tridentata plant tissues and the resulting extracts. By chemically reacting with the ascorbic acid, the toxic NDGA quinones are reduced to NDGA hydroquinone which is NDGA itself.

To concentrate the Larrea tridentata organic extract which is saturated with ascorbic acid, the extract is transferred to a water jacketed, stainless steel tank, which is heated to approximately 100 degrees C. by circulation of a suitable solvent (i.e. water). While the extract is heated in the tank, the organic solvent is boiled out of the extract and may be recovered by condensation for reuse. The extract is heated in the tank to approximately 10 degrees above the boiling point of the organic extraction solvent. This rise in temperature of the extract indicates that the concentrated Larrea tridentata extract is substantially free of the extraction solvent The concentrated extract, approximately 1/10 the volume of the original Larrea tridentata extract, is then removed from the tank and packaged in tightly sealed plastic drums for storage and shipping.

Another aspect of the present invention includes mixing the concentrated Larrea tridentata extract with effective, additional amounts of ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 -mercapto-1,2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol). Formulation of the extract with excess amounts of one or more of the listed reducing agents prevents subsequent formation of NDGA quinone during subsequent processing, storage, and formulating of the extract, and functions as a synergistic antioxidant in vivo with NDGA and other endogenous antiviral compounds.

Our invention is based in part on our discovery that a Larrea tridentata extract, which is produced according to the methods of our invention, contains no detectable amount of NDGA quinone when analyzed by high performance liquid chromatography. Because of the foregoing discussion linking NDGA quinone with the toxic effects associated with the consumption of Larrea tridentata products, the production of Larrea tridentata extract, with a very low concentration of NDGA quinone, for use in medical formulations is very important.

Further, we have found through experiments performed by the inventors, that formulations based on the Larrea tridentata extract and ascorbic acid compound, as described in this invention, have pronounced antiviral activity against Herpes simplex virus types 1 and 2, and against Kaposi's Sarcoma in human patients. We have also found through experimentation that the described formulations have pronounced antiviral activity against Herpes simplex virus type 1 (HSV-1) in both animal cell culture models and human volunteers as well as anti-inflammatory action in human volunteers.

The present invention therefore provides methods for the medically useful and effective extract of Larrea tridentata and formulations for production of pharmaceutical agents which have immediate and commercially important utility in the medical treatment of viral and inflammatory diseases.

Experimental Results

The following are provided by way of illustrating examples of extraction and processing formulations of the Larrea tridentata extract, and are provided by way of illustration only and are not intended to limit the invention in any way.

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EXAMPLE 1

Plant material (consisting of mostly leaves but with some small branches and a small amount of fruits and flowers) was harvested from Larrea tridentata shrubs growing in the Arizona desert, southwest of Gila Bend, Arizona. The plant material was air dried in the shade at ambient temperature and humidity for one week before processing. 100 kg of plant material was extracted with 100 liters of F.C.C. grade acetone by recirculating the acetone over the plant material three times. The resulting Larrea tridentata extract was filtered through Whatman #1 filter paper.

5 mils of F.C.C. grade polysorbate 80 was added to 50 liters of the extract and the extract was slowly passed through a glass column packed with 250 g of powdered ascorbic acid. The extract, saturated with ascorbic acid, was concentrated by boiling off the acetone solvent in a water-jacketed stainless steel tank which was heated to approximately 100 degrees Celsius by circulating hot water. The resulting concentrated extract, in the form of a viscous liquid, was collected in plastic drums and used to prepare medical formulations.

EXAMPLE 2

Several formulations of concentrated Larrea extract suitable for encapsulation were prepared by thoroughly mixing the concentrated extract, as produced in example 1, with dry excipient materials including starch, sucrose, fructose, and ascorbic acid powder. In some cases, other ingredients including antiviral and anti-inflammatory agents or herbal extracts i.e. Echinacea, Podophyllin, etc. were also combined in formulations. In all cases, the mixtures consisted of one part concentrated Larrea tridentata extract with 5 to 10 parts dry excipient material. These formulations were used to treat HIV opportunistic infections, and Herpes virus infections described in this specification. The treatment consisted of administering several capsules containing 50 to 100 mg of Larrea tridentata extract per capsule which were ingested daily.

EXAMPLE 3

Several formulations of concentrated Larrea tridentata extract in a lotion base were prepared by adding the concentrated extract, as produced in example 1, into various lotion formulations. The concentration of extract added to the lotion base can range from 0.1 to 5% volume/volume. In initial trial with human volunteers, a 3% lotion prepared according to the principles of the present invention has been used successfully in the medical treatment of athletes foot, viral lesions caused by herpes simplex virus (HSV-1, HSV-2), Kaposi's Sarcoma, and inflammation of the skin induced by contact allergens, ultraviolet light and thermal exposure. The treatment consisted of applying the lotion frequently and liberally to the affected areas.

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EXAMPLE 4

Results obtained by the inventors show that the concentrated Larrea extract, produced by the methods of the present invention, when used at a concentration greater than 20 micrograms per milliliter, is nearly 100 % effective in protecting African Green Monkey kidney cells from destruction by the Herpes simplex-1 virus (HSV-1).

It is apparent from the evidence presented above that the methods and compositions of the present invention meet long-standing needs in the medical treatment of viral and inflammatory diseases.

Many alternatives to the most preferred embodiment, described above, are also part of the present invention. For example, when the NDGA quinone is reduced using ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 -mercapto-1,2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol) in a Larrea tridentata extract, it is not essential to add further amounts of one of these listed reducing agents as described. While doing so provides a more stable nontoxic product, if additional amounts of one or more of these listed reducing agents are not added, the resulting extract will still have use as a nontoxic therapeutic agent.

Moreover, it is not essential to use any of the listed reducing agents to reduce the NDGA quinone present initially in the extract. If another, perhaps even toxic, reducing agent is used to reduce the NDGA quinone naturally occurring in the Larrea tridentata extract, ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (ie. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 -mercapto-1, 2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol) can then be added to the resulting product to produce a nontoxic extract which will not develop quantities of NDGA quinone through oxidation during processing and storage. In such a process, an additional step to remove the reducing agent would likely be necessary.

Finally, as noted in the prior art NDGA has many known uses as an antioxidant food additive and as a therapeutic agent. NDGA for these purposes can be produced by extraction from natural sources, such as the Larrea tridentata, or can be synthesized. However the NDGA is produced, during processing and storage it oxidizes to produce NDGA quinone and is therefore toxic when ingested. Accordingly, it is within the scope of the invention to combine NDGA, whether extracted or synthesized, with ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (ie. sodium ascorbate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic acid), monothioglycerol (3 -mercapto-1,2-propanediol), potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate, sodium bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E (DL-alpha-tocopherol) to produce a stable, nontoxic NDGA product that can be used as a food additive or therapeutic agent.

The preceding description has been presented only to illustrate and describe the invention. It is not intended to be exhaustive or to limit the invention to any precise form disclosed. Many modifications and variations are possible in light of the above teaching.

The preferred embodiment was chosen and described in order to best explain the principles of the invention and its practical application. The preceding description is intended to enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the following claims.

* * * * *

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