probiotics

Probiotic Digested CulturesProbiotic Digested Cultures for GGT Reduction for GGT Reduction

Functional Nutrition CenterFunctional Nutrition CenterCoeur d’Alene, IdahoCoeur d’Alene, Idaho

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Basic ResearchBasic 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 Systemic Effect of Probiotic Microflora-HistoryMicroflora-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 GMDP Treatment of A549 cells Inhibition of TNF-alpha cytotoxicityInhibition of TNF-alpha cytotoxicity

A549 cells

0

0.5

1

1.5

media no TNF TNF +CHX

1 ug/mlGMDP

2 ug/mlGMDP

4 ug/mlGMDP

LD

H R

ele

as

e

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GMDP Treatment of A549 cells GMDP Treatment of A549 cells Inhibition of Inhibition of Fas mediated cytotoxicityFas mediated cytotoxicity

A549 cells

0

0.1

0.2

0.3

0.4

0.5

control anti FASAntibodies

1 ug/ml GMDP

LD

H A

ctiv

ity,

OD

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

Results of p50 ELISA

NF-κB 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.

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

Raw RLU, Blanked RLUs, Fold increase Treatment 250ng Protein/well 250ng prot/well over treatmentNo treatment 17.453 Blank N/ATNF alpha/CHX 224.696 207.243 12.87GMTP, 2.5 µ/ml 16.227 -1.226 0.93GMTP, 10 µ/ml 22.474 5.021 1.29GMDP, 2.5 µ/ml 11.470 -5.983 0.66GMDP, 10 µ/ml 12.748 -4.705 0.73

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Summary of in vitro data on Summary of in vitro data on probiotic wall fragment GMDPprobiotic 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 GGTP as a main risk factor of all cause mortalityof all cause mortality

Brenner et al. Prev. Med (1997)

0

1

2

3

4

< 15 15 - 19 20 - 29 30 -49 > 50

GGTP Level, U/L

Rel

ativ

e R

isk

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GGT Reduction with GMDP GGT Reduction with GMDP 20 mg daily for 3 weeks20 mg daily for 3 weeks

9 cancer patients with various initial levels of GGTP activity

Groupof 3withavg.

380 U/L

0

50

100

150

200

250

300

350

400

0 3 weeks

GG

TP,

U/L

Groupof 3withavg.

144 U/L

0

50

100

150

200

250

300

350

400

0 3 weeks

GG

TP,

U/L

Groupof 3withavg.

92 U/L

0

50

100

150

200

250

300

350

400

0 3 weeks

GG

TP,

U/L

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Administration of GMDP: Effects on Administration of GMDP: Effects on GGT activityGGT activity

Control

-

25

50

75

100

0 day 14 day

GG

TP

Ac

tiv

ity,

%

GMDP

-

25

50

75

100

0 day 14 day

GG

TP

Ac

tiv

ity,

%

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 Administration of GMDP: Effects on LDH activityLDH activity

Group of 19 / placebo

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25

50

75

100

0 day 14 day

LD

H A

ctiv

ity,

%

Group of 23 / 1.5 mg/kg

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25

50

75

100

0 day 14 day

LD

H A

ctiv

ity,

%

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.

Group of 21 / 0.5-1 mg/kg

-

25

50

75

100

0 day 14 day

LD

H A

ctiv

ity,

%

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Administration of GMDP: Effects on Administration of GMDP: Effects on Triglyceredes and CholesterolTriglyceredes and Cholesterol

placebo

-

25

50

75

100

125

0 day 14 day

Trig

lyce

rid

es,

%

GMDP 1.5 mg/kg

-

25

50

75

100

125

0 day 14 day

Trig

lyce

rid

es,

%

GMDP 0.5-1 mg/kg

-

25

50

75

100

125

0 day 14 day

Trig

lyce

rid

es,

%

placebo

-

25

50

75

100

0 day 14 day

Ch

ole

ster

ol,

%

GMDP 0.5-1 mg/kg

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25

50

75

100

0 day 14 day

Ch

ole

ster

ol,

%

GMDP 1.5 mg/kg

-

25

50

75

100

0 day 14 dayC

ho

lest

ero

l, %

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 Summary of our clinical observationsobservations 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, Summary of GMDP effects, cont.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 Natural GMDP-Product InformationInformation

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 GMDPManufacturing of natural GMDP

Structure of L. plantarum PGN

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PGP from PGP from L. acidophilusL. acidophilus Inhibition of TNF-alpha cytotoxicityInhibition of TNF-alpha cytotoxicity

Cytoprotection by PGP from probiotics

A549human lungcarcinoma

cells

0

0.2

0.4

0.6

0.8

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

LD

H a

ctiv

ity,

OD

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.

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Synergistic effects of Probiotic Synergistic effects of Probiotic Peptidoglycans (PGP) and IsoflavonesPeptidoglycans (PGP) and Isoflavones

A549 cells

0

0.2

0.4

0.6

TNF100_U/ml

Isoflavons1_ug/ml

PGPL.plantarum

1_ug/ml

PGP +Isoflavons1_ug/ml

LD

H A

ctiv

ity,

OD

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Effects of D-aminoacids on TNF Effects of D-aminoacids on TNF alpha cytotoxicityalpha cytotoxicity

A549 cells

0

0.2

0.4

0.6

0.8

TNF100_U/ml

L-glutamine+ NAG1_ug/ml

D-glutamine+ NAG1_ug/ml

GMDP1_ug/ml

LD

H R

elea

se

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Dietary management with PGP from Dietary management with PGP from L. plantarumL. plantarum18 year old patient with aplastic anemia18 year old patient with aplastic anemia

Case study history

– 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

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Treatment with PGP from Treatment with PGP from L. plantarumL. plantarumVery high daily dose of probiotic originVery high daily dose of probiotic origin

Blood CBC. 18 year old patient with aplastic anemia

22-Feb-01 Start PGP 1 g/day

18-Jun-02 low cyclosp & steroids4-Sep-02 off cyclosp & steroids

-

20,000

40,000

60,000

80,000

1-Feb-01 2-May-01 31-Jul-01 29-Oct-01 27-Jan-02

Platelets, Thou/cm

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Treatment with PGP from Treatment with PGP from L. plantarumL. plantarum Very high daily dose (1 g) of probiotic originVery high daily dose (1 g) of probiotic origin

Blood CBC. 18 year old patient with aplastic anemia

-

20,000

40,000

60,000

80,000

1-Feb-01 1-Feb-02

Platelets,Thou/cm

-

1,500

3,000

4,500

6,000

1-Feb-01 1-Feb-02

WBC, thou/cm

-

2

4

6

8

1-Feb-01 1-Feb-02

Neutrophils,Abs.Aut.

-

10

20

30

40

1-Feb-01 1-Feb-02

Hematocrit-

4

8

12

16

1-Feb-01 1-Feb-02

Hb, g/dl

0

1

2

3

4

1-Feb-01 1-Feb-02

RBC, million/cm

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Perspective Clinical ApplicationsPerspective Clinical Applications

Based on established in vitro and in Based on established in vitro and in vivo effectsvivo effects

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Clinical Applications in Clinical Applications in Gastroenterology and EndocrinologyGastroenterology 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 Clinical Applications in CardiologyCardiology

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 MedicineEmergency 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 OncologyClinical 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 NeurologyClinical Applications in Neurology

Prevention and Treatment of Hemorrhagic and Ischemic stroke

Parkinson Disease ALS MS

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Infectious DiseasesInfectious 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