Probiotic Digested Cultures for GGT Reduction

Functional Nutrition Center

Coeur dAlene, Idaho

Basic Research

Identification and expression of the probiotic autolysin genes Preparation of the proprietary glucopeptides after specific autolysin hydrolysis Preclinical testing of their antioxidant and apoptosis modulating activities

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Systemic Effect of Probiotic Microflora-History

Short fragment of bacterial wall (GMDP) was isolated from lysozyme hydrolysate of Lactobacillus Bulgaricus (Blastolysine) in Russia and tested as a powerful immunomodulator with antitumor activity in 1975-1980 GMDP was identified in the human milk and amniotic fluid (PCT Publication WO 08/510,737), 1995 Antibodies to GMDP was identified in the blood serum of 700 healthy people (Pinegin B, et al.,FEBS Letters, 1995).

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Results of In Vitro Studies

Our Data NIH, Japan Abbotts Labs
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GMDP Treatment of A549 cells

Inhibition of TNF-alpha cytotoxicity

1.5

A549 cells

LDH Release

0.5

0 media no TNF TNF + CHX 1 ug/ml 2 ug/ml 4 ug/ml GMDP GMDP GMDP

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GMDP Treatment of A549 cells

Inhibition of Fas mediated cytotoxicity
0.5

LDH Activity, OD

0.4 0.3 0.2 0.1 0 control anti FAS Antibodies

A549 cells

1 ug/ml GMDP

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Effects of GMDP on NF-kappa B expression, Ross Product Div., Abbott Labs

NF-B p50 ELISA assay was done on A549 human lung carcinoma cells. The samples were pools of duplicate nuclear extracts prepared from duplicate plates of treated cells and stored at -80 C. The same amount of protein was added to the plate for each sample, as determined by Bradford protein assay (mean of two assays). All sample dilutions , control, and blank were tested in triplicate wells. The results given are the mean values. The primary antibody dilution was 1/2000 and the conjugate dilution was 1/50K. The values were read on the luminometer with a 1 second integrated reading.
Treatment No treatment TNF alpha/CHX GMTP, 2.5 /ml GMTP, 10 /ml GMDP, 2.5 /ml GMDP, 10 /ml Raw RLU, 250ng Protein/well 17.453 224.696 16.227 22.474 11.470 12.748 Blanked RLUs, 250ng prot/well Blank 207.243 -1.226 5.021 -5.983 -4.705 Fold increase over treatment N/A 12.87 0.93 1.29 0.66 0.73

Assay information: Average RLU for background wells (complete lysis buffer)=4447 Average RLU for positive control wells (Jurkat nuclear extract) minus background=209.546 CHX- cycloheximide RLU- relative light unit

Results of p50 ELISA

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Summary of in vitro data on probiotic wall fragment GMDP

Inhibits negative inotropic effect of TNF alpha Cytokine blocker Inhibits FAS antigen killing pathways Inhibits NF-kappa B expression in cancer cells (Abbott Labs) Inhibits GGTP mRNA expression in cancer cells and after proinflammatory stimulation (Niida S., NIH, Japan)

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Clinical Observations
Institute of Medical Food, Inc & Medical Radiological Center, Russia Anatoly F.Tsyb, MD (Oncology) Rita Ellithorpe, MD (Family Medicine) Julian Whitaker, MD (Cardiology) Alan Sosin, MD (Internal Medicine) Vladimir Slesarev, MD (Oncology)

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GGTP as a main risk factor of all cause mortality

4 Brenner et al. Prev. Med (1997) 3 2 1 0 < 15 15 - 19 20 - 29 30 -49 > 50 GGTP Level, U/L

Relative Risk

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GGT Reduction with GMDP

20 mg daily for 3 weeks
400 400 400

350

300

250

Group of 3 with avg. 380 U/L

GGTP, U/L

350

300

250

Group of 3 with avg. 144 U/L

GGTP, U/L

350

300

250

Group of 3 with avg. 92 U/L

GGTP, U/L

200

200

200

150

150

150

100

100

100

50

50

50

weeks

weeks

weeks

9 cancer patients with various initial levels of GGTP activity

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Administration of GMDP: Effects on GGT activity

Control

GMDP

GGTP Activity, %

GGTP Activity, %

100 75 50 25 0 day 14 day

100 75 50 25 0 day 14 day

7 patient with elevated levels of GGTP activity showed reduction in this activity following treatment with GMDP

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Administration of GMDP: Effects on LDH activity

Group of 19 / placebo
LDH Activity, %

Group of 21 / 0.5-1 mg/kg 100 75 50 25 0 day 14 day

LDH Activity, %

Group of 23 / 1.5 mg/kg 100 75 50 25 0 day 14 day

LDH Activity, %

100 75 50 25 0 day 14 day

GMDP was given orally at a dosage of 0.5-1 mg/kg daily to 21 cancer patients, 1.5 mg/kg to 23 patients, and placebo was given to 19 patients. During the follow-up period, the overall clinical condition of all patients treated with GMDP improved. Nobody experienced any side effects.

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Administration of GMDP: Effects on Triglyceredes and Cholesterol

placebo
GMDP 0.5-1 mg/kg
Triglycerides, % Triglycerides, %

GMDP 1.5 mg/kg 125 100 75 50 25 0 day 14 day

Triglycerides, %

125 100 75 50 25 0 day placebo 14 day

125 100 75 50 25 0 day 14 day

GMDP 0.5-1 mg/kg

Cholesterol, % Cholesterol, %

GMDP 1.5 mg/kg 100 75 50 25 0 day 14 day

Cholesterol, %

100 75 50 25 0 day 14 day

100 75 50 25 0 day 14 day

7 control and 11 treated patients with 2-10x elevated levels of lipids

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Summary of discovered clinical effects of GMDP

Immunostimulation in patients with colorectal cancer (Prof.Morris et al., 1996) Stimulation of WBC

Reduction of ALT and AST

Activation of cytochrome p450 Lowering bilirubin

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Summary of our clinical observations

Reduction of TNF alpha cytotoxicity both in vitro and in vivo Lowering levels of ALT, GGTP, LDH, AST, and Alkaline Phosphatase Lowering Cholesterol Reducing Triglycerides Increasing of Ejection Fraction

Lowering Fe++ Increasing WBC Reducing nausea Increasing thrombocytes Reduction of Congestive edema

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Summary of GMDP effects, cont.

Lowering High Blood Pressure Lowering high blood glucose Drastic improvement in patients with chronic fatigue syndrome

Significant amelioration of cancer and CHF fatigue Increasing low blood pressure Lowering viral load (based on quantitative PCR). Burn pain relief

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Predigested Probiotic Cultures with Natural GMDP

In vitro Clinical Data

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Natural GMDP-Product Information

The product was developed after discovery that hydrolysis of the peptide bonds and peptide cross link of the gram positive bacteria genus of Lactobacillus and Bifidum leads to release of the novel glucosamine muramyl peptides (GMDP), with strong potency towards inhibition of TNF alpha cytotoxicity, following reduction in gamma glutamyl transpeptidase activity (GGTP)The product is completely organic; its preparation is based on a method for isolation of high purity biodegradable glucosaminemuramyl peptides, which comprises the bacterial wall isolation with subsequent lysozyme and endopeptidase (papain) hydrolysis and purification with preparative high pressure liquid chromatography (HPLC).

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Manufacturing of natural GMDP

Structure of L. plantarum PGN

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PGP from L. acidophilus

Inhibition of TNF-alpha cytotoxicity
0.8

0.6
LDH activity, OD

A549 human lung carcinoma cells

0.4

0.2

0 control TNF, 100 u/ml PGP, 1 ug/ml

Lactate dehydrogenase (LDH) is a stable cytosolic enzyme, product of a housekeeping gene that is released upon cell lysis. Released product is measured in rapid enzymatic assay, measuring the conversion of tetrazolium salt into a red formazan product. In our experiments we used LDH release assay in order to asses the TNF induced cell death and consecutively the cytoprotection provided by certain compound to the TNF and FAS induced cytolysis.

Cytoprotection by PGP from probiotics

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Synergistic effects of Probiotic Peptidoglycans (PGP) and Isoflavones

0.6

LDH Activity, OD

A549 cells
0.4

0.2

0 TNF 100_U/ml Isoflavons 1_ug/ml PGP L.plantarum 1_ug/ml PGP + Isoflavons 1_ug/ml

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Effects of D-aminoacids on TNF alpha cytotoxicity

0.8

LDH Release

0.6 0.4 0.2 0 TNF 100_U/ml

A549 cells

L-glutamine D-glutamine + NAG + NAG 1_ug/ml 1_ug/ml

GMDP 1_ug/ml

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Dietary management with PGP from L. plantarum 18 year old patient with aplastic anemia

significant fatigue and prolonged bleeding hemorrhagic petechiae in the skin bone marrow biopsy > Aplastic anemia Epstein-Barr virus high doses of prednizone, neoral, cyclosporine, neupogen, and erithropoietin > condition was steadily deteriorating One year later, started PGP from L. plantarum
Case study history

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Treatment with PGP from L. plantarum

Very high daily dose of probiotic origin
80,000

Platelets, Thou/cm
60,000

40,000

22-Feb-01 Start PGP 1 g/day

20,000

18-Jun-02 low cyclosp & steroids 4-Sep-02 off cyclosp & steroids
2-May-01 31-Jul-01 29-Oct-01 27-Jan-02

1-Feb-01

Blood CBC. 18 year old patient with aplastic anemia

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Treatment with PGP from L. plantarum

Very high daily dose (1 g) of probiotic origin
80,000 6,000 4 60,000 4,500 3

40,000

3,000

20,000

Platelets, Thou/cm
1-Feb-02

1,500

WBC, thou/cm
1-Feb-01 1-Feb-02 0 1-Feb-01

RBC, million/cm
1-Feb-02

1-Feb-01

16

40

12

30

Neutrophils, Abs.Aut.

20

10

Hb, g/dl
1-Feb-01 1-Feb-02 1-Feb-01

Hematocrit
1-Feb-02 1-Feb-01 1-Feb-02

Blood CBC. 18 year old patient with aplastic anemia

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Perspective Clinical Applications

Based on established in vitro and in vivo effects

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Clinical Applications in Gastroenterology and Endocrinology

Diabetes (both type I and type II) and its complications. Hepatitis C and B Liver fibrosis and cirrhosis Fat liver Metabolic syndrome

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Clinical Applications in Cardiology

Reduction of GGTP for the prevention of secondary heart attack and cardiovascular mortality Chronic Heart Failure Hypertension (works as a calcium channel blocker) Hypertriglyceremia Atherosclerosis

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Emergency Medicine

Septic shock Cardiogenic shock Trauma shock Burn shock Tumor shock Pancreanecrosis Ischemic-reperfusion injury Detoxification (drugs, alcohol, and narcotics) Pregnancy toxicity (hypertension and eclampsia)

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Clinical Applications in Oncology

Treatment and Prevention of Glioblastoma Treatment and Prevention of Metastasis of: Breast carcinoma Colorectal cancer Ovarian carcinoma Lung cancer Liver cancer Melanoma Postchemotherapy leucopenia and thrombocytopenia

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Clinical Applications in Neurology

Prevention and Treatment of Hemorrhagic and Ischemic stroke Parkinson Disease ALS MS

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Infectious Diseases
HIV HCV HPV Herpes Epstein-Barr virus Tuberculosis

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Effects to be taken into account

Calcium Channel Blocker Anticoagulation Effect Lowering Blood Glucose Lowering High Blood Pressure

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