BACKGROUND OF THE INVENTION

Living multicellular organisms synthesize proteins composed of only L-amino acids. D-

amino acids occur in bacterial peptides. In the early antibiotic era, Lipmann demonstrated that the

small peptides tyrocidine and gramicidin contained D-amino acids (Lipmann, F., Hotchkiss. R.D.,

Dubos, R.J. J. Biol Chem. 141 :163, 1941.) Peptidoglycan (synonym: murein) is a another D- amino acid containing a net-like molecule that surrounds and confines the bacterial cell (Andronova, T., Ivanov, V. Sov. Med. Rev. Immunol. 4: 1-63, 1991.) It is the stress-bearing and

form-giving component of bacterial cell wall. Peptidoglycan consists of a backbone of alternating

strings of N-acetylglucosamine and N-acetyl-muramic acid interconnected by short D-peptides.

This basic structure is found in the cell wall of nearly all bacteria, including Lactobacillus

Bulgaricus, Bifidum, and Streptococcus Thermophilous (Andronova et al, Link, Harriet. Dietary Immunostimulator European Patent 0 432 490, Sasaki, et al. Japan Patent #62265231). These

bacteria are found to be useful in preventing gastroenteritis in infants (Saavedra, J., Bauman, N.,

Oung, I., Perman, J.A., Yolken, R.H. Lancet 344: 1046-49, 1994.)

Ellouz et al. identified muramyl dipeptide (i.e., N-acetyl-D-muramyl-L-alanyl-d- isoglutamine) as the rmnimal structure capable of replacing the mycobacterial component of Freud's complete adjuvant (Ellouz, et al. Biochem. Biophys. Res. Comm. 59:1317-1325, 1974.)

Since this discovery, the synthesis of MDP has allowed study of many of the biological aspects of

adjuvants, such as immune modulation, increase of the resistance to infection and the production of fever. The many biologic properties of GMDP and MDP give promise of a wide range of

potential clinical applications such as vaccine adjuvants, non-specific anti-infection agents, anti¬

cancer agents and treatment of immunosuppressed status. However, because of its impressive

ability to induce fever (pyrogenicity), MDP itself did not prove suitable for broad clinical

development (Krueger, J.. et al, Somnogenic Compositions and Method of Use. US patent

4,698,330.) Fortunately, many structural analogs of MDP have been synthesized, several of

which appear to have vigorous immunostimulatory properties with minimal somnogenic and pyrogenic side effects (Andronova et al).

One-hundred percent prevention of the post-weaning diarrhea in the piglets by orally

administering of peptidoglycans derived from Bifidum bacteria has been demonstrated by Sasaki

et al. They have found that peptidoglycans stimulates the IgA secretion in the mucosa and decreases the number of Salmonella bacteria in the intestine. Their method was inefficient when

the active component is used during first 5 days after birth. Lately, Link and Pahud have isolated

MDP from Lactobacillus Bulgaricus and proposed to administer it as dietary immune stimulator

(European Patent #0 432 490).

N-acetyl-glycocyamine-muramyl dipeptide (GMDP) was isolated during analysis of the anti-tumor drug, blastolysine, which is a lysozyme cell wall hydrolysate of Lactobacillus

Bulgaricus (U.S. Patent No. 4,395,399). This compound has been extensively studied in animals, demonstrating adjuvant activity, antitumor activity, low pyrogenicity and hypnogenic effect (Andronova et al). The presence of MDP in human urine, has been reported by Krueger

(Krueger, J.M., et al. J. Biol. Chem. 257:1664-1669, 1982.) Chemical and physiological properties of this urinary factor resemble those of the sleep factor found in sterile cerebrospinal

fluid and in acid/aceton extracts of brains from sleep-deprived animals (Garcia-Arraras and Pappenheimer. J. Neurophysiol. 49:528-533, 1983.) In a accordance with this discovery, biological compositions for the induction of the sleep have been invented (U.S. Patent No. 4,698,330). It has been suggested that muramyl peptides enter the organism as a result of

adsoφtion of degradation products of normal colibacilli. Numerous reports testify to the fact that

muramyl peptides induce different mediators of the immune response, such as interleukin- 1 (IL-1) and Ia- antigen, both in immunocompetent cells and in brain astrocytes (Dinarello, C.A., and

Krueger, J.M. Fed. Proc. 45:2545-2548, 1986, Vermeulen, M.V. J. Immunol. (1987), 139:7-9. Thus, muramyl peptides maintain the immune status of the organism at a normal level and

promote the normal duration of sleep by means of their specific mediators whose main function is

tightly coupled with the immune system. Similar to some vitamins, muramyl D-peptides are utilized (but not synthesized) by the host organism, and they act as regulators of various

physiological systems.

Human milk glycoconjugates can inhibit replication of a range of pathogenic

microorganisms. Examples include mucin-associated glycoprotein that inhibits rotavirus replication and prevents experimental gastroenteritis (Yolken, R.H., et al. J. Clin. Invest. 90:1984-1991, 1992.) A fucosylated oligosaccharide isolated from human milk inhibits the ability

of stable toxin of E. coli to produce diarrhea in vivo (Newburg, D.S., et al. J. Infect. Dis.

7:1075-1080, 1990.)

In late 1994, investigators at Johns Jopkins reported a significant reduction in the

incidence of acute diarrhea and rotavirus shedding in infants aged 5 to 24 months using an infant formula supplemented with bacteria (Saavedra, J., Bauman, N., Oung, I., Perman, J.A., Yolken, R.H. Lancet 344:1046-49, 1994.) In this study, Bifidobacterin (which constitutes the

predominant intestinal flora of breast fed infants) and Streptococcus thermophilus (a lactic acid

producing bacteria that generates lactase activity) were added to infant formula in at attempt to

prevent rotavirus diarrhea. This successful reduction in the incidence of rotavirus diarrhea using such supplemented formula supports previous uncontrolled studies on the effect of bifidobacteria on the incidence of diarrhea in infants (Tasovatz, B., et al Ann. De Pediatrie. 22:291-297,

1962.) How and why the addition of "bacteria" to infant formula causes a decrease in acute

diarrhea and rotavirus shedding remains unknown.

BRIEF DESCRIPTION OF THE INVENTION

Applicants suggest that the "active ingredient" in both bifidobacteria and Streptococcus

hemophilus is a component of the bacterial cell wall, D-peptidoglycans. Furthermore, Applicants

suggest that the peptidoglycans clusters (GMDP) discovered in the human milk are also

responsible for the protective effect of the breast feeding. They are also the strong biological immunomodulating agents, which physiologically stimulate the maturation of the

immunocompetent cells in the newborns. The protocol for the identification of the bacterial

peptidoglycans in human milk and commercially available cultured yogurt is described in examples

1 and 2 of this application. Monoclonal anti-idiotypic antibodies to glucosaminyl-

muramylpeptides have been used in Applicants' technology.

Applicants have predicted the presence of bacterial D-amino acid containing compounds (GMDP) in human milk. Based on this, one aspect of the present invention comprises the

supplementation of infant formula with synthetic GMDP and/or MDP to treat and prevent infection in infants. Such supplementation is believed to modulate the immune systems of infants.

Another aspect of the present invention is to provide a nonspecifically active vaccine,

incorporating in its peptide fragment D-amino-acid in a first, second or third position from the end. Nonspecificity of the vaccination is achieved by exploiting newly discovered phenomena, namely inhibition of binding infectious receptor to its host cell counterpart by D-amino acid containing compounds and increased production of secretory IgA in mucosa organs.

Yet another aspect of the present invention is a nonspecific method for detecting,

preventing, and inhibiting precancer conditions in human beings by vaccination with D-amino-acid vaccines. The monitoring of such non-specific anti-cancer D-vaccine is provided by combined (NMR and ultrasound) in vitro tests on blood serum or peripheral lymph.

BRIEF DESCRIPTION OF THE DRAWINGS

For further details, reference is made to the discussion which follows, in light of the

accompanying drawings, wherein

Fig. 1 shows the results of an assay for GMDP in human milk. It is the adsorption of

Eg/1.2 antibody on the adsorbed GDMP-Lys-PAA (5μg/well).

Fig. 2 shows the results of another assay for GDMP in human milk. It is the adsoφtion of

E6/1.2 antibody on the adsorbed human female milk (lOOμl of 1:2 dilution of milk/well).

Figs. 3A and 3B show competitive inhibition of the adsoφtion of E6/1.2 antibody on the

adsorbed GMDP-Lys-PAA by GMDP in solution. Fig. 3 A: lμG GMDP-Lys-PAA per well; Fig.

3B: 0. lμg GMDP-Lys-PAA per well.

Fig. 4 shows competitive inhibition of the adsoφtion of E6/1.2 antibody on the adsorbed GMDP-Lys-PAA by serial dilutions of yogurt in PBS.

Fig. 5 shows the change in ΔT1 of peripheral lymph after treatment with GMDP.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, useful D-aminoacid containing compounds are muramyl peptides which conform to the general formula:

wherein:

X ¹ and X ² are Cl and C5 acyl groups such as acetyl

R represents the residue of aminoacid or linear peptides built up of from 2 to 8 amino acid residues, at least one of residues being optionally substituted with D-isomer analogue.

The preferred position of this D-aminoacid residue is second or third from the proximal

end, or second or third from the distal end of the linear peptide.

The most preferable position is second from the proximal end.

R preferably represents a di-or tri-peptide residue. The proximal residue is preferably that

of L-amino acid and is selected from the group comprising:

L-alanyl

L-valyl

L-leucyl

L-isoleucyl

L-seryl

L-aminobutyryl

L- threonyl

L-tryptophanyl

L-lysyl

L-ornithyl

L-argynyl

L-histidil

L-ornithyl

L-cysteinyl

L-phenylalanyl

L-tyrosyl

L-arginyl

L-asparaginyl

L-prolyl

L-hydroxyprolyl

L-glutaminyl L-aspartyl L-methionyl and wherein L-alanyl is most preferred.

The next amino acid from the proximal end of the peptide is preferably of the D- configuration. Most preferable are D-isoglutamine and D-glutamate.

L-alanyl and L-lysyl are preferred for a third amino acid position from the proximal end of the peptide.

Most preferred class of D-amino acid containing compounds for use in this invention is:

wherein:

R is a an amino acid or linear peptide built of from 2 to 8 amino acid residues. One of

them is being optionally substituted with a lipophilic group.

The most preferred dipeptides are L-Ala-D-isoGln and L-Ala-D-Glu.

One of the most preferred compounds in this invention which corresponds to the above

formula is N-acetyl-D-glucosaminyl-(l-4)-Nacetylmuramyl-L-alanyl-D-isog lutamine (GMDP) and N-acetyl-D-glucosaminyl-(l -4)-N-acetylmuramyl-L-alanyl-D-glutamic acid (GMDP- A).

Other useful compounds which fall within the above formula II include:

N-acetyl-D-glucosaminyl-(l-4)-N-acetylmuramyl-L-alanyl-D- glutamine-n-butyl-ester (GMDP-OBu)

N-acetyl-D-glucosaminyl-(l-4)-N-acetylmutamyl-L-alanyl-D- isoglutaminyl-L-lysine

(GMDP-Lys)

N- Acetyl-D-glucosaminyl-( 1 -4)-N-acetylmuramyl- N-methyl -L-alanyl -D-isoglutamine

N- Acetyl-D-glucosaminyl-( 1 -4)-N-acetylmuramyl-L-alanyl-D-isoglutamine- 1 -adamantyl ester

L-Threonyl-N-[N-Acetyl-D-glucosaminyl]-L-lysyl-D-prolyl-L -arginine

Those compounds which are members of the most preferred class are N-acetyl-D-

glucosaminylmuramyl-L-alanyl-D-isoglutamine, N-acetyl-D-glusaminylmuramyl-L-alanyl-D-glut- amine and N-acetylmuramyl-L-alanyl-D-glutamine.

The effectiveness of these compounds in prevention of bacterial diarrhea is shown by 100% successful food vaccination of domestic animals. In particular, these D-amino acid

containing GMDP have been shown by appHcants to provide full protection against clostridia and

salmonella diarrhea in piglets by increasing secretion IgA in intestinum mucosa. The effective dosage is in the range of 0.2-0.5 mg/kg for GMDP and 0.5-5.0 mg/kg for GMDP(A). The vaccination effect is dependent upon the dosage regimen and it is believed that this should consist of divided dosages. The protective effect has been demonstrated to last for 5-6 weeks after 3

consecutive administrations.

The aspect of the present invention relating to the pharmaceutical composition varies with

the mode of administration, which may be topical, oral, vaginal, rectal, and as a food supplement.

For topical administration, these vaccine comprise an effective amount of compound of

this class in admixture with pharmaceutically acceptable nontoxic carriers. A suitable range of

composition would be 10%-30% active ingredient. The concentration of D-compound in pharmaceutical compositions suitable for topical appUcation will vary depending upon the

particular activity used in conjunction with the condition and subject to be treated. Suitable

caπies include creams, ointments, lotions, emulsions and solutions.

For oral administration, these compositions contain an effective amount of a D-compound

of this class incoφorated in a mixture with any of the usually employed excipients. such as, for example, pharmaceuticals of mannitol, lactose, starch, talcum, cellulose, glucose, or sucrose. The active ingredient comprises 65%-95% of such formulations. Such vaccine take form of solutions,

tablets, pills, capsules, sustained release formulations and the like.

Vaginal and rectal administration includes the use of Hquids which can be sprayed on the mucosa, or solid suppositories which can be inserted one or more times per day.

In the following specific examples, the results of standard bioassays are described.

Example 1. TESTING OF HUMAN MILK FOR THE PRESENCE OF GMDP.

Antibody capture assay (Antibodies: A laboratory Manual (E. Harlow and D. Lane, eds.), Cold Spring Harbor Laboratory, 1988) has been used to detect GMDP in human female milk. Highly specific mouse E6/1.2 anti-GMDP monoclonal antibody (Ka = 2x10^ M"l) and GMDP

conjugated to polyacrylamide backbone through amino group of lysine (GMDP-Lys-PAA) were

obtained from Dr. Nesmeyanov, the Shemyakin and Ovchinnikov Institute of Bioorganic

Chemistry, Russian Academy of Sciences, Moscow, Russia.

Briefly, GMDP-Lys-PAA or serial dilutions of human female milk in phosphate buffered

saline (PBS) were adsorbed onto wells of microtiter plates for several hours. After washing with

PBS, remaining binding sites were blocked by lh incubations with 200 μl PBS +3% bovine serum

albumin (BSA). After washing with PBS 100 μl of E6/1.2 antibody, diluted 1:1,000 or 1:2,000

with PBS+1%BSA, were added. The incubation was carried out for lh at room temperature. Plates were washed with PBS and incubated for lh at room temperature with 100 μl/well of

1:1000 dilution of goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) in

PBS+1%BSA. After washing 100 μl of OPD solution (1 mg/ml) in 20 mM sodium citrate buffer, pH 4.5, containing 0.03% H2O2 were added. The reaction was stopped 10-20 min later by

adding 50 μl well of 2.5 M H2SO4. Optical density at 492 nm was measured.

The results of the experiments are shown in Figs. 1 and 2. The specificity of the interaction was determined by comparing the adsoφtion of E6/1.2 on milk, adsorbed onto wells of microtiter plates, with the absoφtion of mouse monoclonal IgGl (k) and mouse IgG. From Fig. 2, it is evident that the difference in the absoφtion between E6/1.2 antibody and monoclonal

IgGl(k) is almost threefold and therefore it is specific.

Example 2. TESTING OF COMMERCIALLY AVAILABLE YOGHURT FOR THE PRESENCE OF GMDP.

Antibody capture assay with the antigen competition variation (Antibodies: A Laboratory Manual. (E. Harlow and D. Lane, eds.), Cold Spring Harbor Laboratory, 1988) has been used to detect GMDP in yoghurt (Mountain High Original Style plain yoghurt produced by Meadow Gold

Dairies, Inc., Inglewood, Co; ingredients: cultured milk, non-fat milk, cream, tapioca, pectin, active cultures (L. bulgaricus, S. thermophilus, L. acidophilus and B. bifidum.)).

Two different amounts of GMDP-Lys-PAA in PBS were adsorbed onto wells of microtiter plates (1 μg/well and 0.1 μg/well). Washing and blocking of remaining binding sites

were performed as described above. Before adding to wells, 50 μl of antibody (E6/1.2m, IgGl(k)

or mouse IgG) were incubated with 50 μl of an antigen (GMDP in PBS or series dilutions of

yoghurt in PBS) for 15 min. Further steps were as described above. Inhibition index (I,%) was

calculated from: I = (l-A _o /Aι)xl00, where A ₀ and Ai are optical densities of samples without

and with inhibitor, respectively.

Figs. 3 A and B show the inhibition of E6/1.2 antibody interaction with the adsorbed

BMDP-Lys-PAA by GMDP itself. Figure 4 shows that yogurt competitively inhibits the

interaction of E6/1.2 antibody with GMDP-Lys-PAA.

Example 3. GMDP (A) IN THE PREVENTION C.PERFRINGES DIARRHEA IN

PIGLETS.

A total of 60 mg of GMDP(A) was given to each of 3 piglets (16 weeks old).

GMDP was given orally for 3 consecutive days by administering 20 mg of active

compound diluted in the water.

After 5 days, 85 ml of water with Clostridia Perfringes (10 unit per ml) was given to all

piglets.

None of the vaccinated piglets developed diarrhea, while two control piglets developed

severe diarrhea.

All piglets were sacrificed. Histologic samples were obtained from the stomach,

duodenum, jejunum, and ileum, and stored at 80°C and then PLP fixed.

Indirect antibody fluorescence method was used to detect IgA cells. Anti pig IgA sheep

IgG serum used as the first serum, which served as antigen for anti sheep IgG rabbit IgG second

serum. To decrease the nonspecific component of this reaction, fluorochrome conjugate was diluted 400 times.

Applicants' cell counts were based on individually defined "mucosa tissue units" constituting of 6-μ-thick and 500-μ-wide blocks of tissue.

To compare mucosal specimens, Applicants took into account the localization of selected

tissue unit (base of vilh). The four units counted are derived from corresponding areas in the neighboring sections; when the ceU numbers are smaU, as far as possible two units in each section were included, or enumerations in two similar specimens from the same piglet were combined.

Example 4. INHIBITION HIV gpl20 BINDING TO ITS CD4 RECEPTOR.

Microtiter wells were coated with recombinant, soluble CD4 peptide corresponding the gpl 20 binding domain. GMDP were added to the soHd phase SD4 along with 1 ng of recombinant HIV gp-120. Following incubation overnight at 4°C, unbound reagents were

removed by washing. The amount of gp 120 bound to CD4 was measured by sequential reactions with peroxidase-labeled monoclonal antibody to gpl 20 (2h, 4°C), H2θ2-o-phenylene diamine peroxidase substrate (30 min, 4°C), acidification, and measurement of the antigen-antibody

reaction at 490 nm. Inhibition percentage of this binding was calculated with the following

formula: inhibition = 1 - [(ODsam - OD bl)/(ODgpl20 - ODbl)]*100

where OD sam is optical density of the sample tested, OD gpl 20 is the mean optical density of wells in which gp 120 was tested without added inhibitor, and ODbl is the optical

density of CD4-coated wells reacted with anti-gpl20 antibody in the absence of either gpl 20 or

inhibitor.

TABLE 2. Inhibition of gp 120 Binding to SD4 Receptor by GMDP

Buffer How Through % Inhibition GMDP

< 5 < 5 96

Example 5: COMBINED (SONIC AND NMR) DIAGNOSTIC TEST OF HUMAN PERIPHERAL LYMPH AND BLOOD SERUM.

MATERIALS AND METHODS

The NMR relaxometry of the peripheral lymph was performed in 50 patients 41-74 years

old. Clinical X-ray examinations have revealed a cancer of the lung, stomach, rectum, prostate or mammary gland in 35 of the patients. In the rest of them (15), chronic inflammation of those

organs was diagnosed. Two patients with chronic bronchitis and 3 with gastritis have been

treated by GMDP for up to 6 months, 50 mg GMDP was administered 8 times per month. In respect of the local spreading of the tumor, the patients were distributed in the following way: Tl

4 patients; T2 6 patients, T3 9 patients; T4 6 patients. The lymph was drawn from peripheral lymphatic vessels through the catheter and collected in a receptacle attached to the leg.

Relaxation time was measured using a Minispec PC-20 relaxometer (Bruker, Reinstetten) at a

resonance frequency of 19.8 mHz and a specimen temperature of 39±1°C. Spin-lattice relaxation

time was calculated using the inversion recovery pulse series. To evaluate the water phase of proteins in detail, the samples of peripheral lymph (0.1-0.3 ml) were placed for 20 minutes in a

cell for ultrasound treatment (lW/cm ² ).

STA TISTICAL METHODS

In vitro reproducibility was determined by calculating the means and standard deviations of differences between first and second measurement of lymph samples. A Mann-Whimey test

was used to compare the mean difference of Tl times of the lymph samples.

RESULTS

The standard deviation of the differences of the two consecutive measurements has

demonstrated a satisfactory in vitro reproducibility for Tl (2,4%). According to AppHcants' results, the average Tl value of peripheral lymph from the patients with nontumor diseases was

2.60s, as contrasted with Tl=2.85 for cancer patients. However, this difference was statistically

insignificant with P>0.05. In all patients Applicants have observed the decreasing of Tl values of the lymph after ultrasonic radiation (Tables 3 and 4).

TABLE 3. Tl AND ΔT1 OF PERIPHERAL LYMPH IN CONTROL PATIENTS

Disease Number of Tl ΔT1 Patients M±δ M±δ

Chron. Pneumonia 6 2.64 ± 0.188 0.078 ± 0.038

Ulcer of Stomach and Duodenum 4 2.45 ± 0.25 0.096 ± 0.011

Adenoma of Prostate 3 2.46 ± 0.356 0.03 ± 0.036

Benign Tumor of Lung 1 2.78 0.31

Cyst of Mammae 1 2.90 0.50

15 2.60 ± 0.253 0.11 ± 0.112

TABLE 4. Tl AND ΔTl IN CANCER PATIENTS

Location of Number of Tl ΔTl Mahgnant Tumor Patients M±δ M±δ

Lung 11 2.83 ± 0.072 0.18 ± 0.008

Stomach 7 2.84 ±0.104 0.225 ± 0.09

Rectum 6 2.88 ± 0.092 0.166 ± 0.044

Uterus 4 2.84 ± 0.084 0.135 ± 0.04

Prostate 3 2.92 ± 0.116 0.276 ± 0.098

Uterus* 4* 2.78 ± 0.05 0.10 ± 0.014

35** 2.85 ± 0.083 0.191 ± 0.067

* Patients with lymphedema after treatment

** Value of Tl and ΔTl without Tl of patients with lymphedema

The parameter Tl (the difference between Tl values measured before and after ultrasound treatment of the peripheral lymph) was higher than 0.12 s in all cancer patients. Based on this

parameter, Applicants observed statistically significant mean differences between control (n=15)

and cancer patients (n=35). Fig. 5 represents the change of ΔTl of peripheral lymph after

treatment with GMDP. ΔTl has been diminished by 30% and has dropped from 0.09 sec to 0.06

sec.

STATEMENT OF UTILITY

The method of the present invention is useful in the prevention and remediation of disease

in humans and domestic animals.

It will be understood by those skilled in the art that the present invention has been desribed

with reference to specific examples but other variations are possible without departing from the

inventive concept. Accordingly, it is desired that the scope of the invention be determined only with reference to the appended claims.