WO1997010845A1 - An immunostimulant and antineoplastic glycan obtained from mycobacterium vaccae - Google Patents

Abstract

The present invention is a glycan compound with antineoplastic and immunostimulant properties for treating human cancers and as an adjuvant in the formulation of a tuberculosis vaccine, said compound containing at least one component from the class of sugars including arabinose, mannose, glucose and galactose, together with a peptide side chain, having an approximate 60-65 kDa molecular weight, and being readily water-soluble and heat-stable. The invention also is comprised of a process for extracting the glycan compound from the Mycobacterium vaccae, a non-pathogenic, rapid growing member of the mycobacteria.

Description

AN IMMUNOSTIMULANT AND ANTINEOPLASTIC GLYCAN OBTAINED FROM MYCOBACTERIUM VACCAE

BACKGROUND OF THE INVENTION

This application claims priority from U.S. provisional application no. 60/003,892, filed on September 18, 1995.

Field of the Invention

This invention relates specifically to a complex polysaccharides and, more particularly, to immunostimulant and antineoplastic compounds and processes for isolating such compounds from a non-pathogenic rapid growing member of the Mycobacteria, Mycobacterium vaccae.

Summary of the Related Art

Knowledge of the inverse relationship between bacterial infection, especially tuberculosis, and cancer can be traced back nearly two hundred years. From 1898 onwards Coley noted the anticancerous activity of mixed microbial infections on humans and Pearle in 1929 reviewed literature back to the turn of the 19th century in which the apparent anticancer activity associates with tuberculosis infections was evident. In a recent review of the envelope of Mycobacteria, Brennan and Nikaido (1995) made the case that the exocellular and surface-exposed materials in Mycobacterium tuberculosis were mostly polysaccharides, specifically D-glucans, D-arabino-D-mannans and D- mannans, but no biological activity was associated with these materials by the authors. However, in 1978 Suzuki et al. reported finding an arabinomannan (SSM) in the Aoyama B virulent strain of Mycobacterium tuberculosis. This material, identical with or similar to Z-100 of the Japanese company Zeria Pharmaceutical Ltd., has apparently been tested against animals since its isolation. However, since this material is isolated from a dangerous and virulent microorganism there are advantages to be gained in determining if similar materials can be isolated from avirulent members of the Mycobacteria. In the process of our exploration of Mycobacteria! glycans with antineoplastic the testing of PS4 in vitro against confluent Caco-2 human adenocarcinoma cells; and, Example 8 describes the in vivo testing of PS4 as a solution injected into the flank or as enteric coated granules given orally against Caco- 2 cells growing in the flank of nude mice. The organism, Mycobacterium vaccae, strain no TMC 1526, obtained from the American Type Culture Collection (Rockville, MD) as Number ATCC 15483, was grown on tryptic soy broth (Gibco Laboratories, Detroit, MI) in culture flasks. After three days at 37 "C, the cells were collected and washed with double distilled water by centrifugation and extracted by boiling with water, 20 mL per mg wet cell weight under reflux for two hours. After allowing to cool, the extract was filtered through a 0.45 μm pore diameter membrane filter (Nalge Co. , Rochester, NY) and dialyzed through Spectra/Por 2 tubing with a molecular weight cut off of 12-14 kDa (Spectrum-Med Ind., Los Angeles, CA) against distilled water overnight. The crude extract was then concentrated 400-800 fold using a rotary evaporator under reduced pressure. At this stage the extract was passed through an LH20 column and the tubes corresponding to the main peak collected. This material was lyophilized to provide a white to off-white powder termed PS4. After this separation, a second stage purification was carried out by passing PS4 through a Sephadex G-75 column to yield a component termed PS4A.

This product was formulated in sieved hydroxy-propylmethylce- llulose (HPMC) granules, particle diameter approximately 400-500 μm diameter by adding PS4 (20% w/w), mixing by dampening with a water spray and redrying. The dried granules were then coated with an enteric coating using Eudragit™-S100 by a methylene chloride in-water solvent evaporation technique described by Ciftci and Groves (1995). In this method the core granules (containing drug) were suspended in a Eudragit solution 7% in methylene chloride. This solution was emulsified in aqueous polyvinyl alcohol solution (0.35% w/v) and the solution agitated until the methylene chloride had evaporated. The granules were collected and dried before Fig. 2 is a graphic representation of the non-specific sugar profile as the compound of the present invention is passed down a Sephadex LH-20 column.

Fig. 3 is a graphic representation of the non-specific sugar profile as the purified extract identified as the compound of the present invention is passed through a Sephadex G-75 column.

Fig. 4 is a diagrammatic view of the effect of pH on the drug delivery system employed to investigate the compound of the present invention and related method for isolating it. Fig. 5 is a graphic representation of the effects produced on growing Caco-2 tumor cells implanted into the blanks of nude mice by both direct subcutaneous injection of a sterile solution of the compound of the present invention or bolus oral administration of the granules containing the compound of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The novel compound described in detail below comprises a glycan, hereafter termed "PS4", and was isolated from a culture of Mycobac¬ terium vaccae. PS4 is used as a therapeutic agent in the treatment of human cancers and as an adjuvant component in the formulation of a tuberculosis vaccine.

The following examples illustrate practice of the invention by which a complex polysaccharide or glycan can be extracted and purified from cultures of Mycobacterium vaccae. More specifically, Example 1 illustrates the extraction process; Example 2 illustrates the purification process; Example 3 describes the formulation of a sterile solution of PS4; Example 4 formula¬ tion of PS4 in an enteric coated hydroxypropylmethylcellulose granule for oral administration; Example 5 is an account of structural characterization of the extract termed PS4 carried out to date; Example 6 describes the activity testing of PS4 against a murine SI 80 sarcoma in mice; Example 7 describes - 6 -

EXAMPLE 2

The material obtained under Example 1 was purified by reconstitution in distilled water and passed through a column of Sephadex LH- 20 (Pharmacia Fine Chemical, Piscataway, NJ). As fractions were removed from the column, sugars were non-specifically determined using the phenol - sulfuric acid method of Hodge and Hof reiter (1962) and are seen to be concentrated between fraction numbers 18 and 24, Fig. 2. Further purifica¬ tion was achieved by passing the material collected between fraction numbers 18 and 24 and passing this through a column of Sephadex G-75 (Pharmacia Fine Chemical, Piscataway, NJ), collecting the material of the single peak,

Fig. 3, between fraction numbers 18 and 26. This material, lyophilized to a white powder, was identified as PS4A.

EXAMPLE 3

The material identified as PS4 in the process outlined in Example 1 was dissolved, 10 μg/mL, in phosphate-buffered saline and sterilized by passage through a sterilized 0.2 μm pore diameter membrane filter (Acrodisc, Gelman Sciences, Ann Arbor, MI) into a presterilized elastomeric-stoppered glass vial under aseptic conditions. This sterile solution was stored at 4-5 ^*C prior to use.

EXAMPLE 4

Hydroxypropylmethylcellulose (HPMC) (Methocel K100M,

Dow Chemical Co., Midland, MI) was mixed with 20% w/w dried PS4

(Example 1) and granulated by mixing and spraying with distilled water prior to drying. The dried granules were sieved to provide granules approximately 400-500 μm in diameter.

Acrylic acid copolymer, Eudragit™ S-100 (Rohm Pharmaceutic¬ als, Maiden, MA) was dissolved in methylene chloride and the HPMC granules added before dispersed in water containing 0.7% polyvinyl alcohol sieving. These coated granules have been demonstrated to pass rapidly down the gastrointestinal (GI) tract of the rat, thereafter slowly passing through the colorectal region. This is believed to be due to the pH-sensitive hydration of the Eudragit, allowing the mucoadhesive properties of the HPMC to be exerted when the pH of the GI tract exceeds 6.5. At this point it seems likely that Eudragit will have dissolved away, allowing the HMPC to become hydrated. Drug release using 5-fiuorouracil as a model has been demonstrated to occur mainly in the colorectal region (Ciftci and Groves, loc. cit.).

The examples which follow are for illustrative purposes only and are not intended in any way to limit the scope of the invention.

EXAMPLE 1

Fig. 1 depicts the separation process used to prepare PS4. (1) The lyophilized organism, obtained as Culture No. ATCC 1526 from the American Type Culture Collection, Rockville, MD was distributed into sterile tryptic soy medium (Gibco Laboratories, Detroit, MI) in sterile stoppered flasks and incubated for three days at 37 "C. (2) The cultured cells were collected by centrifugation and (3) washed twice with sterile double distilled water before (4) extracting with boiling water (240 mL per 300 mg wet cell weight) under reflux for two hours. After cooling (5), the extract was filtered (6) through a 0.45 μm pore size membrane filter

(Nalge Co., Rochester, NY). The filtered extract was then dialyzed (7) against distilled water ovemight through Spectra Por 2 dialysis tubing, molecular weight cutoff 12-14 kDa, Spectrum Md. Ind., Los Angeles, CA. The retentate was then concentrated (8) to about 10 mL using a rotary evaporator (Valley Electromag-netics Corp., Spring Valley, IL). Finally (9) this extract was lyophilized using a Labconco Lyph-Lock 4.5 Freeze Drying System (Labconco Corp. , Kansas City, MO) to yield a dry white or yellowish- white powder. - 8

Tice^® substrain BCG Connaught BCG

(PS1A1) (PS1A2) (CFA2) glucose 100% arabinose 38.6 27.5 mannose 18.6 11.7 galactose 7.0 2.5 glucose 35.9 41.7

+ 16.6% of an as yet unidentified sugar residue

The fact that none of the BCG glycans have associated protein or peptide residues strongly proves that PS4 is indeed a new composition of matter.

The protein residue was removed by digestion with pronase, a non-specific protease enzyme, and associated proteins by extraction into phenol. Unexpectedly, we have found that biological activity (SI 80 sarcoma, see Example 6) is diminished by pronase treatment but enhanced by phenol extraction, indicating an intimate structure/activity relationship involving the protein-poly saccharide linkage of PS4.

EXAMPLE 6

This glycan, like others isolated from Mycobacterial species (Lou et al. 1994; Wang et al. 1995) has antineoplastic activity which may be measured in a quantitative fashion using the murine SI 80 sarcoma model. Eight-week old female CFW Swiss Webster mice were injected subcutaneous¬ ly into the right flank with 4.8-4800 μg/kg doses of PS4 in a volume of 0.1 mL phosphate buffered saline and an equal volume of phosphate buffered saline containing 3 x 10⁵ viable SI 80 sarcoma cells. Fourteen days after inoculation the mice were sacrificed and dissected in order to access the incidence of tumors. Intra assay differences between the test and control (Sigma Chemical Co., St. Louis, MO). The coarse emulsion system was stirred at 500 rpm at room temperature until the methylene chloride had evaporated. The granules were collected on a filter, washed with distilled water and dried at room temperature by spreading on a flat surface before storing over a desiccant at 4-5 'C prior to use.

EXAMPLE 5

Purified PS4 (Example 2) is a white powder, readily soluble in water, soluble in methanol and slightly soluble in ethanol or acetone, has an approximate molecular weight of 60-65 kDa by SDS-PAGE and consistently provides one spot by thin layer chromatographic techniques. However, these spots stain for both sugars and protein, indicating that a protein or peptide fragment is closely associated with the polysaccharide component. This has been confirmed by proton NMR studies. When PS4 is hydrolyzed with mild acid and the sugar fragments passed down, a calibrated HPLC column with an amperometic detector (Dionex, Inc.), the composition is approximately:

% w/w arabinose 23.6 mannose 33.1 galactose 26.1 glucose 17.2 This suggests that the main structural element is a galactoarabi- nomannan which appears to be uniquely different from the three biologically active complex glycans isolated from two M. bovis BCG substrains, namely: - 10 -

In vivo, 4-6 week old female athymic nu/nu mice were inoculated subcutaneously (sc) in the right flank with 1 x IO⁷ Caco-2 cells suspended in 0.2 mL of DMEM medium. After 28 days, the mice were randomized into control and treatment groups, 7 mice/group. The control group was injected sc with 0.2 mL culture medium. PS4 doses for sc administration were determined by analogy with doses required for other glycans isolated in this laboratory (Lou et al. , 1994) at 1 and 10 μg/mouse respectively. Oral doses, again at 1 and 10 μg/mouse, in Eudragit-coated HPMC granules were given by gavage. Tumor growth was estimated by palpation and measurement with calipers twice weekly over the following 42 days. Tumor volume, v, was estimated as v = 4/3 x x r, x r₂ x r₃, where r,, r₂ and r₃ represent length, width and height, respectively. Data were treated by analysis of variance and Student's test using a Microsoft-Excel software program and are shown in Fig. 5. These data illustrate the marked inhibition of tumor growth rate at 10 μg PS4/mouse and the suppression of growth at 1 μg. This effect is a typical dose/response often seen with immunostimulant glycans and is consistent with the failure of the material to exert any biological effect in vitro. These materials, of which PS4 is a unique example, are acting as immunostimulants, apparently (without being bound by theory) stimulating phagocytes to produce cytokines such as interferon-γ and interleukin-2 which enhance the antiinflammatory effects due to these cells.

EXAMPLE 8

The compositions of the present invention are used to treat a variety of neoplastic diseases including but not limited to sarcoma as discussed in Example 6, melanoma, lymphoma, plasmocytoma, leukemia, carcinomas such as renal cell carcinoma, carcinoma of the lung, breast carcinoma, colon carcinoma, prostate carcinoma and others. Typically, the composition is administered as a pharmaceutical composition comprising the glycan-peptide of the present invention in combination with a pharmaceutically acceptable groups at 14 days were tested for significance by Fisher's Exact Test. Tests run on several occasions provided approximate specific activities for different glycans, with one unit of activity defined as a titer of the lowest quantity of assayed material causing a significant inhibition of tumor incidence (see Lou et al. 1994 for details) as follows: u/mg PSI (crude) 316 (Lou et al. , 1994)

PS1A1 > 10^s (unpublished data)

PS1A2 10⁵

CFA2 3 x 10⁵ PS4 > IO⁵

PS4 (phenol extraction) 15,873

PS4 (pronase treatment) < 100

Thus, of the four glycans identified to date with significant antineoplastic activity (as measured by the SI 80 murine sarcoma model), PS4 is approximately equally active on a weight basis compared to the glycans isolated from the two BCG materials.

EXAMPLE 7 ln vitro activity was tested by exposing Caco-2 human colorectal adenoma cells to both solutions and the HPMC coated formulation of PS4. The cells were plated (63 x 10³ cells/cm²) onto rat tail collagen- coated Transwell tissue culture inserts (3 μm pore size). The cells were maintained with culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 % non-essential amino acids and 0.01 % penicillin-streptomycin (from Gibco Laboratories, Grand Island, NY)). Incubation at 37 "C was in a 5% CO₂/95% relative humidity atmosphere was carried out, and confluence was determined by microscopic examination and the measurement of the transepithelial electrical resistance. Doses of PS4 ranged from 0.6 to 60 μg/mL from both solution or from formulation but no effect on the cell confluence was detected by either evaluation method. - 12 - antigen may be combined with the composition of the present invention in an oil-based carrier, a water soluble carrier or any other pharmaceutically acceptable carrier, adjuvant, diluent or excipient in which both the target antigen and the composition are stable. Among their many uses, such combinations are useful in new immunotherapeutic strategies directed against Mycobacterial diseases such as tuberculosis. In such combinations, the composition of the present invention is useful for stimulating a host immune response to target antigens derived from or related to antigens found associated with Mycobacterium tuberculosis. Such combinations are also be useful in treating neoplastic disease. Also, the composition itself serves as an immunotherapeutic for the treatment or prevention of Mycobacterial diseases.

The foregoing illustrative examples relate to a specific glycan or series of glycans isolated from the non-pathogenic rapid growing Mycobac- terium vaccae. While the present invention is described in terms of a specific microorganism and a specific process for isolation of a specific glycan, it is understood that variations and modifications will occur to those skilled in the art upon consideration of the present invention.

For example, it is entirely possible that other mycobacteria will produce an identical or substantially similar glycan to PS4 which will have the potential commercial advantages that we see with M. vaccae. In addition, we have described an extraction and purification process that we have found to be of general application to the extraction and purification of other glycans from other mycobacterial organisms. It is also feasible that other extraction processes using lower or higher temperatures, other aqueous miscible solvents such as acetone, methanol, ethanol, isopropanol, on polyethylene glycols or non-aqueous solvents such as hexane may have improved utility. Classical separation processes used for the commercial isolation of high molecular weight polymeric sugars such as dextrans from growth media of organisms carrier, adjuvant, diluent or excipient. The glycan is administered subcutane¬ ously, intradermally, intramuscularly, intravenously, intravaginally, rectally, orally, intraperitoneally or by other well known routes of administration.

Determining appropriate dosages and routes of administration of the compositions is readily determined by routine methods. The endpoints for the determination of proper dosages necessary to achieve a therapeutic effect may include a significant increase in survival time over untreated control patients, a significant decrease in tumor size or volume, diminution or prevention of metastases and/or the reversal or amelioration of physiologic symptoms of neoplastic diseases, for example, diminution or reversal of cachexia.

The compositions of the present invention may also be adminis¬ tered alone or with other anti-neoplastic agents including chemotherapeutic agents or with immunotherapeutic agents to provide either additive or synergistic therapeutic effects.

For example, the compositions of the present invention may be co-administered with immunotherapeutic agents such as those described by

Kim et al. , Cancer Immunol. Immunother., 38: 185-193 (1994); Cohen, E.

PCT/US95/05794; Porgador et al , Int. J. Cancer, 53:471-477 (1993); Rosenberg et al , J. Clin. Oncol., 10: 180-199 (1992) and several others.

The composition may also be administered as an adjuvant to traditional chemotherapeutic regimens either before, during or after the chemotherapeutic regimen is undertaken.

EXAMPLE 9 The compositions of the present invention are also used as adjuvants to potentiate the immune response to a particular target antigen or antigens. This may be accomplished by mixing a preparation of the target antigen to which an immune response is desired with the composition of the present invention before administration to a recipient animal. The target - 14 -

References cited.

All of the references cited in the specification and below are incorporated herein by reference.

Brennan, P.J. and Nikaido H. The envelope of Mycobacteria. Ann. Rev. Biochem., £4:29-63 (1995).

Lou, Y. et al. Initial characterization of an antineoplastic polysaccharide-rich extract of Mycobacterium bovis BCG Tice^® substrain. Anticancer Res. , 14: 1469-1476 (1994).

Wang, R. et al. An antineoplastic glycan isolated from Mycobacterium bovis (BCG vaccine). Biochem. J. (in press).

Suzuki, I. et al. Effect of SSM, an extract from human type tubercle on the granulopectic activity of the reticuloendothelial system. II. Activation of carbon-phagocytic function and its dose-response. Pharmacometr- ics, 1&79-85 (1978).

Ciftci, K. and Groves, M.J. The in vitro and in vivo activity of an enteric coated drug delivery system containing 5-aminofluoracil against Caco-2 tumors, paper in press (1995).

Hodge, J.E. and Hofreiter, B.T. Phenol sulfuric acid colorimetric method. Methods in Carbohydrate Chemistry, 1:388-389 (1962).

Kim et al , Cancer Immunol. Immunother. , 28: 185-193 (1994).

Cohen, E., PCT/US95/05794

Porgador et al , Int. J. Cancer, 52:471-477 (1993).

Rosenberg et al , J. Clin Oncol., IQ: 180-199 (1992). such as Leuconostoc mesenteroides may also have utility in the initial isolation of PS4 from M. vaccae cultures in a large scale production process.

- 16 -

8. A method for preparing an antineoplastic and/or an immunostimulatory composition comprising a glycan in association with a peptide from a culture Mycobacterium comprising the steps of: a) growing a culture of Mycobacteria under suitable nutrient conditions; b) collecting said culture of Mycobacteria; c) boiling said collected Mycobacteria in water under reflux thereby producing an extract; d) filtering said extract; e) dialyzing said extract thereby producing a retentate; f) concentrating said retentate; g) lyophilizing said retentate; h) reconstituting said retentate; i) chromatographing said reconstituted retentate through a Sephadex LH-20 column; j) isolating fractions from step i) which contain sugars; k) combining said isolated fractions isolated in step j);

1) chromatographing said combined fractions obtained in step k) over a Sephadex G-75 column; m) collecting fractions from step 1) which contain sugars; and n) lyophilizing said fractions collected in step m).

9. The method of claim 8 wherein said Mycobacteria is Mycobacterium vaccae.

Claims

I CLAIM:

1. A composition comprising in combination; a glycan in association with a peptide, said composition having a molecular weight of about 60 kDa to about 65 kDa, wherein said composition is derivable from Mycobacterium.

2. The composition of claim 1 wherein said Mycobacterium is Mycobacterium vaccae.

3. The composition of claim 1 wherein said glycan is a galactoarabinomannan.

4. The composition of claim 3 wherein said galactoarabino¬ mannan comprises about 23.6% w/w arabinose, about 33.1 % w/w mannose, about 26% w/w galactose, about 17.2% glucose.

5. The use of the composition of claim 1 in the manufac- ture of a medicament useful as an immunostimulant and for the treatment of neoplastic disease, wherein said composition comprises a galactoarabinomann¬ an in association with a polypeptide, said composition being derivable from Mycobacterium.

6. The use of claim 4 wherein said Mycobacteria is Mycobacterium vaccae.

7. The use of claim 5 wherein said galactoarabinomannan comprises about 23.6% w/w arabinose, about 33.1 % w/w mannose, about 26.1 % w/w galactose and about 17.2% w/w glucose. 10. The method of claim 8 wherein said glycan is a galactoarabinomannan .

11. The method of claim 10 wherein said galactoarabinoma¬ nnan comprises about 23.6% w/w arabinose, about 33.1 % w/w mannose, about 26.1 % w/w galactose and about 17.2% glucose.

12. The method of claim 8 wherein said composition has a molecular weight of about 60 kDa to about 65 kDa.

13. A composition produced by the method of claim 8.

14. A method for treating neoplastic diseases comprising administering to a subject mammal having a neoplastic disease, an antineoplas- tically effective dose of a composition according to claim 1 or 7.

15. A vaccine useful in the treatment or prevention of tuberculosis comprising administering to a host a therapeutically effective dose of a composition according to claim 1 or 7.

16. A vaccine according to claim 15 further comprising antigens derived from Mycobacterium tuberculosis, said antigens capable of inducing an immune response in a host.

17. A pharmaceutical composition comprising a composition according to claim 1 or 7 in combination with a pharmaceutically acceptable adjuvant, diluent, carrier or excipient.

18. The pharmaceutical composition of claim 17 further comprising an enterically coated tablet.