The present invention relates to new complexes of the peptidoglycan monomer (PGM) with trivalent metals, to a process for the manufacture thereof and to their use as immunomodulators in pharmaceutical preparations.

It has been known, that upon submersed culturing of the bacteriae Brevibacterium divarucatum (NRRL 2311) and Micrococcus glutamicus (ATCC 13287) in the presence of an inhibitor of the cell wall biosynthesis there are excreted peptidoglycan fragments, from which there is upon incubation with lysozyme and fractiυnatiυn on molecular sieves isolated the well defined peptidoglycan monomer (PGM), N-acetyl- glucosaminyl-N-acetyl-muramoyl-L-alanyl-D-isoglutaminyl-(L)- n eso- diaminopimelyl-(D)-amide)-(L)-D-alanyl-alanine (Yug. patent 40,472, AT 362,740; Carbohydr. Res.110 (1982), 320-325), of the formula I

CO-NH- CH- CO-NH-CH- CONH, I I ^3 -

CO-NH-C I H-CO- H-C ' H-CO-NH-C I H-COOH

(CH ₂ ) ₃ CHa CH ₃

CH-NH ₂

I

CONH,

which demonstrates an immunostimulating and antimetastatic activity (Z. Immun.- Forsch. 155 (1979), 312-318; Eur. J. Cancer Oncol. 19 (1983), 681-686). It has been shown by our earlier investigations, that under exactly defined conditions PGM forms complexes with bivalent metals (Yug. pat appl. P-1982/86) of a highly complex structure, identified according to analytical and spectral data as a basically chelate structure, which is also characteristic for some amino acids or peptides, (Acta Pharm. Jug. 38 (1988), 310; J. Serb. Chem. Soc 55, (5) (1990), 265-269). The investigations, performed by M. Tonkovic et al (Inorganic Chim. Acta 161 (1989), 81-85) demonstrated, that PGM forms in aqueous solution in excess of Fe(HI) ions and without correction of pH of the solution a complex with Fe(IH) ions of a completely different structure. In the continuation of our work on said problems, further investigations were performed in the complexing of PGM with other trivalent metals under various conditions.

Surprisingly, it has now been found, that some trivalent metals yield novel formations with PGM in solution and at a specific pH. Thus, there were evidenced upon potentiometric titration such effects for aluminum /A1(HI)/, rhodium/CHI)/, lanthanum /La(IH)/, and vanadium V(IH)/, whereas, no such effects have been detected for iron /Fe(m)/, and bismuth /Bi(HI)/. (Fig.1 - 4).

In the enclosed Fig.1 - 4, "A" stands for 0.01M PGM (25 mL), "C stands for "A + B", whereas, "B" stands in Fig.1 for 0.005M Al ³⁺ , in Fig.2 for 0.005M Rh ⁺ , in Fig.3 for 0.005M La ³⁺ , and in Fig.4 for 0.005M N ³⁺ .

It was established by potentiometric titration data that the formed complexes possess different stability constants, significantly increased for the aluminum complex / PGM-A1 (HI) - K / as well as the vanadium-complex /PGM-N(III)-K/ in comparison with those values for bivalent metals (Table 2). That means a possibility for much better pharmacokinetic characteristics of the trivalent metal ions complexes. It is evident as well, that trivalent metals form at various pH values in solution two different complexes (Table 1). Only those at higher pH values for each metal are subject of this invention.

There can be also noticed a significant difference in the preparation of our trivalent metal complexes in comparison with the investigations of M. Tonkovic et al.

Namely, under conditions described in this invention Fe(Iϋ) as well as Bi(Iϋ) ions did not form any complexes with PGM.

Calculation:

[ Lo ] .α[ L ] n=

[Mo ]

K ₂ = ή-1 2 > ή > 1

(2-ή)[L]

K _S = K _1X K ₂

TABLE 1 - Survey of complexes with trivalent metal ions

TABLE 2- Comparison with other metal complexes of PGM

A further object of the present invention is a process for the preparation of complexes of the peptidoglycan monomer with trivalent metals, such as e.g. aluminum, lanthanum, vanadium and rhodium (except iron and bismuth), wherein the peptidoglycan monomer is reacted in aqueous solution with a corresponding trivalent metal salt upon correction of the pH of the solution with alkalies, and the product is isolated by concentration of the obtained solution to a reduced volume, at a pH of about 4.2 for the aluminum salt, of about 7.5 for the lanthanum salt, of about 8.0 for the rhodium salt, and of about 5.0 for the vanadium salt, followed by the addition of an organic solvent which is miscible with water, yet is a non-solvent for the complex, and finally the obtained product is isolated by filtration.

The hydrolysis of such isolated complexes in 6N hydrochloric acid yielded upon the amino acid analysis: alanine, glutamic acid and diaminopimelic acid in a ratio of 3:1:1, which proves that the peptide composition was retained in the complex, in accordance with PGM. By this process there were found sugar components, which themselves were partially decomposed during hydrolysis. C NMR spectra also confirm the presence of all fragments, which constitute PGM. However, some signals are significantly shifted (Table 3), which demonstrates the coordination of the metal with the PGM molecule.

Table 3. Characteristic shifts of the ¹³ C NMR spectra for complexes of PGM with rhodium and lanthanum

*B.Klai<5 Carbohydr. Res.110 (1982), 320-325

In PGM-Rh(HI)-K, for example, there are significantly shifted the signals for C atoms of the terminal alanine, furtheron some C atoms of the diaminopimelic moiety of the molecule, which demonstrates the coordination with the amino group, and the new spatial arrangement of said moiety of the molecule. In the muramine moiety of the molecule there disappears the signal for the C atom of the beta-anomeric conformation. This instructs on the formation of a coordinating bond, forming such a configuration of the complex, that for the OH" group in position C _j of the muramine moiety no arrangement at position β is possible. On the contrary, in PGM-La(HI)-K there are present signals for the alpha and beta anomer, yet they are shifted towards the lower values for approximately 10 ppm (90 and 85 ppm), which leads to the conclusion that lanthanum coordinates with this moiety of the molecule upon formation of a new configuration of the complex, different from those with Me(II) ions.

The increased stability of the complex in comparison with PGM per se in alkaline medium, is also of utmost importance for the biological approach to the use of the complex. The rhodium complex, for example, is stable at a pH of 8, whereas, PGM is definitely decomposed in alkaline medium. It is also important, that blood serum contains enzymes which very rapidly break up the PGM into the sugar and the peptide components. Some metal ions, especially magnesium, have a noticeable role therein. In the absence of magnesium said enzymes are not effective. (J. TomaSic et al, J. Chromatogr. 440 (1988), 405 - 414). In a complex, where the metal site is already occupied, the enzyme is not effective and the molecule may not be decomposed for a longer time. The presence of metals may not only significantly effect the stability and the biotransformation, but also the distribution and the elimination, as well as other properties of the molecule. Biological tests of some complexes of PGM with trivalent metals were performed by employing the standard Jernej method of PFC testing on CBA mice (female). The investigation was performed by employing the standard peptidoglycan monomer (PGM) and the physiological solution (FO) as blank controls. The results are listed in Table 4, and demonstrate the equal effectiveness of the examined complexes and the standard PGM in the stimulation of the immunological response of the receivers.

Table 4. Immunomodulating activity of the complex of PGM with trivalent metals

According to the known and established Prior Art there have not been described complexes of PGM with trivalent metals with the exception of iron (HI), for compounds of the peptidoglycan structure.

The invention is illustrated by the following Examples.

Example 1. Preparation of the complex of the peptidoglycan monomer with aluminum

The peptidoglycan monomer (504.50 mg, 0.50 mmole) is dissolved in 25 mL of water and upon addition of Al( θ3)3 x 9H2O (93.75 mg, 0.25 mmole) there is kept stirring for 1 hour. The pH of the solution is then adjusted to 4.2 by the addition of 0.1N NaOH and stirred for one additional hour. The mixture is concentrated by evaporation under reduced pressure to a volume of about 2 mL, and within about 1/2 hour there is added dropwise 50 mL of acetone through a separating funnel. A white precipitate is obtained, which is separated by filtration, thoroughly aspirated, washed with ethanol, and dried under reduced pressure to constant weight The yield 460 mg.

ffiKBR (cm- ¹ ): 3400 - 3260, 3070, 2970, 2920, 1650, 1535, 1380, 1350, 1040, 595 M.p.=200 °C (decomp.).

UV _max (H ₂ 0) (nm)=192, A ^1% =608.42

Metal analysis by means of atomic absorption spectrophotometry.

Example 2. Preparation of the complex of the peptidoglycan monomer with rhodium

In accordance with the process, as described in Example 1, with the sole exception that rhodium trichloride trihydrate (RI1CI3 x 3 H O, 65.83 mg, 0.25 mmole) is charged instead of aluminum nitrate, and then aqueous sodium hydroxide is added up to a pH of 8.0 and the solution is stirred for 2 additional hours. Yield 490 mg.

IRKBR (cm" ¹ ): 3370 -3270, 3070, 2980, 2915, 1650, 1535, 1450, 1370, 1315, 1230, 1160, 1105, 1065, 1040, 535. M.p.=200 °C (decomposition).

UNmax (H2O) (nm): 192.440 (shoulder); A ^1% (192 nm) = 608.42.

Example 3. Preparation of the complex of the peptidoglycan monomer with lanthanum

In accordance with the process, as described in Example 1, with the sole exception, that aCl3 x 7 H2O (92.85 mg, 0.25 mmole) is charged instead of aluminum nitrate, and then aqueous sodium hydroxide is added up to a pH of 7.5. Yield 479 mg.

_IR KBR ( _cm -l). 3400 - 3240, 3060, 2980, 2915, 1650, 1550, 1425, 1410, 1370, 1315, 1245, 1160, 1110, 1070 - 1040, 600. M.p.=200 °C (decomposition).

UN _max (H ₂ O) (nm): 190; A ^1% (190 nm) = 576.47.

Example 4. Preparation of the complex of the peptidoglycan monomer with vanadium

In accordance with the process, as described in Example 1, with the sole exception, that VCI3 (3933 mg, 0.25 mmole) is charged instead of aluminum nitrate, and then aqueous sodium hydroxide is added up to a pH of 5.0. Yield: 455 mg.

I _R KBR (cm" ¹ ): 3400 - 3260, 2940, 1670 - 1650, 1550, 1460, 1380, 1320, 1260 - 1235, 1120, 1062, 1050, 625

UV _max (H ₂ O) (nm): 194; A ^1% (194 nm) = 294.54.