Hydrogen- Clinical use and future prospects

In this presentation we will show how hydrogen can have a positive effect on:

Cancer Inflammation/pain killer

Diabetes Atopic dermatitis

The Problem

Every single day we are exposed to the harmful effect of our environment. Pollution, sun exposure and various toxins from a broad range of sources damage our cells. Science is starting to understand the biology behind this damage. One of the main reasons is caused by the molecules known as ‘free radicals’ or Reactive Oxidative Species (ROS). Typically, molecules have two electrons to keep them stable. The free radicals, however, only have one electron due to the harmful environmental impact, which makes them unstable and a contributing factor to the breakdown of our bodies.

The unstable free radical are always seeking to become stabilised by pulling in an additional electron from healthy molecules. This process sets off a chain reaction and more molecules become imbalanced and thus unhealthy. A damaging process known as oxidative stress. Persistent oxidative stress is one of the major causes of most lifestyle-related diseases, cancer and the ageing process.

Everyday we are exposed to cell-damaging free radicals

The Solution

Antioxidants are substances that protect your cells from oxidative stress and inflammation by removing the harmful free radicals. The amount of free radicals in the body must be in balance to maintain a good health. Certain vitamins are known as antioxidants and are believed to counteract oxidative stress by reducing the formation of free radicals. Nevertheless, vitamins and nutrients are linked to a limited therapeutic success. On the other hand, molecular hydrogen has potential as a novel antioxidant in preventative and therapeutic applications.

Hydrogen readily gives up electrons to ROS, which stabilises them and stops them in their tracks. Hydrogen shows not only effects against oxidative stress, but also various anti-inflammatory and anti-allergic effects. Hydrogen holds advantages over other antioxidants because it is such a tiny molecule: it is able to penetrate cell membranes, and can easily enter deep into cell components, where other antioxidants are not able to reach. Also, hydrogen boosts the body’s defences by increasing production of natural antioxidants. No adverse effects of consuming hydrogen exist.

Molecular Hydrogen eliminates free radicals.

ROS and their potential implications

ROS

DNA Oxidation

Protein oxidationLipid oxidation

Cancer

Cell ageing

Tissue ageing/dedegeneration

Proliferation / metastasis

Disease/ageing

Intracellular organ degeneration

Oxidative deterioration

AGE production

Inflammation promotion

Immune system cell growth

Osteoclasts promote

Intracellular progression

Acidification Enzyme Synthesis

Growth factor stimulus

Physiological ActivityCytotoxic Action

ROS

Two types of ROS

Hydrogen as a selective antioxidant

Selective, clean, and safe. While taking high doses of supplemental antioxidants has negative health effects, it is impossible to take too much H2. Molecular Hydrogen ONLY scavenges and neutralises the harmful free radicals, leaving the beneficial ones alone. Many antioxidants leave behind toxic byproducts, but the only byproduct of H2 is water.

Therapeutic target

Physiological activity: Low

Cytotoxic activity: high

TROSToxic ROS

Adverse effects due to removal

Target selectivity is important for antioxidant therapy

BROSBiological ROS

stimulationStrong

weak

ROS activity and physiological activity/cytotoxicity

Scientific?The wide-ranging therapeutic and preventative benefits of molecular hydrogen have been evidenced in over 850 peer-reviewed, scientific articles, encompassing over 170 disease models including: type 2 diabetes, rheumatoid arthritis and other autoimmune diseases, metabolic syndrome, Parkinson’s & Alzheimer’s disease, heart disease, numerous forms of cancer

Major papers/clinical trials since 2007

Immune activation / inflammation and ROS

• Block TNFα induction in the NFκB pathwa• NO suppression by inhibition of iNOS activity• Suppress IL-6 mRNA expression• Suppression of ICAM-1 mRNA expression• Maintaining mitochondrial function

TNFα NFκB

ROS

Cytokine

COX

Cytotoxicity

iNOS

TNFα

2013: hydrogen molecule suppresses TNFα induced cell damage

2014: Effect of inhalation of hydrogen gas after cardiac arrest

· Suppression of decline in behaviour/cognitive function.· Neuronal cell death/inflammatory response decreased markedly.

By hydrogen inhalation

· Neuronal cell necrosis decreases.· Suppress hippocampal / cerebral cortex microglial activity.

2015: Hydrogen drip suppressed intestinal mucosal damage due to hemorrhagic shock

· Administration of hydrogen to the ischemic injury of the digestive tract mucosa caused by hemorrhagic shock· Histologically, reduction of intestinal villous damage, reduction of neutrophil infiltration· Reduction of MDA, MPO, IL-6, TNFα (increase of inflammatory cytokines) in the hydrogen administration group· Increase in SOD, IL-10 (antiinflammatory cytokine / increase in antioxidant substance) in hydrogen administration group

Appendix 2016: Onset of Parkinson's disease by mutation of DJ-1 gene

DJ-1

O2

Mitochondria

ROS

Cell/DNA

ROS

AntioxidantDJ-1

O2

Mitochondria

ROS

Cell/DNA

ROS

Antioxidant

Oxidative damage

Oxidative damage: Acquired Genetic mutation: Familiar

SignalStress

H2?

2016: Multi-center double-blind study on Parkinson's disease

Confirm effectiveness with large-scale double-blind trial conducted on Parkinson's disease• H 2 Hydrogen water containing 5 mM 1 L / day 8 weeks

Evaluation:• UPDRS (unified Parkinson 's disease rating scale)

Confirmed effect in: Hydrogen administration can inhibit the progression of Parkinson's disease

Clinical UseFrom basic medicine to clinical medicine

Exploring administration methods considered from previous papers

1: Dissolved in liquid before administration

2: Gas administered, dissolving in the body

* Water (digestive tube) * drip * injection *

* Inhalation (lung) * Dermal Injection*

3: Generated in the gastrointestinal tract, dissolved in the body

* Hydrogen body occlusion (oral administration) * Acceleration of development by intestinal bacteria (Acarbose etc.)

Drip Injection

Infusion/injection solution Hydrogenated physiological saline

Gas concentration in cylinder Filled gas concentration Humidity Filling pressure

: >99.999% : 99.9999% : 0% : 0~100kPa

Hydrogen dissolution

• Henry's law • Boyle/Charles' law • Intermolecular force of hydrogen gas • Intermolecular force of liquid • Presence in the nano bubble state

(Ppm measurement)

Dissolution limit ≒1.57ppm

Dissolved hydrogen concentration in drip bag

0.0

0.6

1.2

1.8

2.4

3.0

Fill straight up Immediately after Shake After 5 minutes After 10 minutes After 15 minutes After 20 minutes After 50 minutes After 60 minutes After 24 hours After 48 hours

0

2.1

1.6

1.8

2.4

2.2

1.9

1.71.6

1.4

0

1.9

1.51.6

2.1

1.7

1.5

1.31.2

1.1

0

1.4

1.21.3

1.5

1.3

1

0.7

0.50.4

Atmospheric pressure filling shake 10 seconds 90kpa filling - Shake 10 seconds 90 kpa filling - shake 15 seconds

ppm Dissolution limit (1.57 ppm) hyper phenomena due to pressurised dissolution

Water drip

Hydrogen gas injection

Perform various inspections before and after

What is 8-OHdG?Steady-state levels of DNA damages represent the balance between formation and repair. Average frequencies of steady state endogenous DNA damages in mammalian cells. The most frequent oxidative DNA damage normally present in DNA is 8-oxo-dG, occurring at an average frequency of 2,400 per cell. When 8-oxo-dG is induced by a DNA damaging agent it is rapidly repaired.

Increased levels of 8-oxo-dG in a tissue can serve as a biomarker of oxidative stress.

Oxidative DNA damage, such as 8-oxo-dG, likely contributes to carcinogenesis (cancer)

The contribution happens via two different mechanisms. The first mechanism involves modulation of gene expression, whereas the second is through the induction of mutations.Thus 8-oxo-dG, if not repaired, can directly cause frequent mutations, some of which may contribute to cancer.

Oxidative stress and 8-OHdG

8-OHdG 8-hydroxy-2’-deoxyguanosine

Normal deoxyguanosine (dG)

HO

Oxidative stress

Urine

Elimination by repairing enzymes

This process is constantly occurring tens of thousands of times per cell/day

· Genetic damage is 5 to 500,000 per day (× 60 trillion) per cell· Detect damage due to oxidation· 8-OHdG test ≈ oxidative stress speed to genes

Relationship between administration and 8-OHdG

OH DNAdamage

repair

ApoptosisCell death / destruction

Cell ageing

Cancer

8-OHdG

Nuclear membrane

Cell membrane

H2O

H2

H2

H2

In the blood

Interstitial

in the cell

In the nucleus

Reduction of 8-OHdG by administration≈ nuclear migration of hydrogen molecules≈ Protection action of DNA

Regeneration by stem cells

Changes of 8-OHdG by hydrogenated physiological saline infusion

Reference value4~13

• 32 patients treated with hydrogen saline infusion• In nearly all cases, a decrease in 8-OHdG is observed• Braking the oxidation damage speed of DNA

Changes in TARC due to hydrogen drip infusion

Changes in TARC due to infusion

0

750

1500

2250

3000

Start End

Patient A Patient B Column 1

<450pg/ml 450~700pg/ml

>700pg/ml

:Normal :Mild :Moderate or higher

TARC (Thymus and activation-regulated chemokine) It is a type of chemokine with leukocyte chemotaxis, and when overproduced, it is believed that Th2 cells are attracted to the lesion locus, production of IgE antibody and activation of eosinophils occur, and an allergic inflammatory reaction is caused. Specificity is seen in atopic dermatitis, it increases markedly as severe, and it decreases with lightening.

Currently accumulating more data

Injection

Hydrogen (nano bubble) injection and its possibilities

Intramuscular injection Tendon sheath injection

Intra-articular injection

Action of physiological saline Research as an analgesic mechanism of trigger point from previous study Used for pain relief at the Study Meeting of Fascial Pain Syndrome (MPS) Fascial exfoliation with physiological saline is analgesic The analgesic effect is stronger for hydrogen (bubble) water

Dissolved water (water molecules) role Suppression of hydrogen NFκB?

Action of hydrogen nano bubbles The action varies depending on the type of gas in the bubble There is no announcement of hydrogen nano bubbles Anti-inflammatory effects of nano bubbles? Other action? Possibility of unknown action

NFκB

TNFα

OH COX Inflammation

Cytokine/iNOS

Acute irritation

Nano bubble action?Other action?

Adhesion peeling action

Consideration of intra-articular injection· Shoulder periarthritis· Osteoarthritis of the knee· Rheumatoid arthritis· Hip acetabular dysplasia· Ankle sprain sequelae· Temporomandibular disorders

Intra-articular injection

Pain reliefReduction of pain during motion/walkingReduction of joint swellingExpansion of range of motion

Age-related changeAthleteCollagen diseaseAfter trauma etc.

Oral Hydrogen Administration

Hydrogen Water

• Water containing molecular hydrogen • Ingested as "hydrogen" • Hydrogen molecular weight that can be administered is small • Hydrogen intake of 11.5 cc / l as 1.5 ppm upper limit • It is unstable and easy to come off

Hydrogen Capsules

• Substances that generates molecular hydrogen in the body • MgH 2, flanagan, coral calcium etc. exist • The most amount generated per gram is MgH 2 • The theory of generation (chemical formula) is clear: MgH 2 • Amount generated per hour> is unknown (fluctuation) • MgH 2 is prescribed as "research reagent" because food is unauthorized

Carbohydrate absorption retarder

• Encourage the generation of hydrogen gas by intestinal bacteria • There is a paper bonacarbose • Different amounts depending on intestinal bacterial load/type

Types of oral hydrogen administration

What are AGEs?Advanced glycation end products (AGEs) are proteins or lipids that become glycated as a result of exposure to sugars. They can be a factor in ageing and in the development or worsening of many degenerative diseases, such as diabetes, atherosclerosis, chronic kidney disease, and Alzheimer's disease

Changes in subcutaneous AGEs by long-term oral administrationAGEs

1.5

1.8

2

2.3

2.5

13.6,3 13.10.2913.11.2613.12.31 14.1.28 14.2.25 14.3.15 14.4.22 14.6.2 14.6.24 14.7.15 14.8.15 14.11.15

AGEs

Hydrogen Gas Inhalation

H2

H2

H2

H2

H2

H2 H2

Inhalation H2 concentrationTemperature (body temperature)

Suction pressureContact area

1 60% O2 21% Is necessary 37℃ 1 α CPAP/Positive pressure breathing etc. The alveolar area about 70m2

H 2 concentration in blood/interstitial fluid/intracellular fluidWhat is the saturation limit?How long will it take to reach the limit?

Body Fluid Volume of body weight: 60% Intracellular fluid: 40%Tissue liquid: 15%Plasma · Lymph fluid: 4.5%Cavity fluid: 0.5%

Alveolus of inhaled hydrogen gas → vascular migration

H2

H2

Interstitial

Intracellular

Hydrogen inhalation: still at the research stage

Exhalation ppm

0

100

200

300

400

Before inhalation 3 min 10 min 20 min 30 min

ppm Hydrogen concentration in expired gas

Inhalation for 60 minutes 3 minutes After atmospheric spontaneous breathing

Suction gas composition: Hydrogen: 60% Oxygen: 30% Water vapor: 10% + Fresh air

It is at present unknown whether it is free from blood or residual hydrogen of dead space

Hydrogen Gas Injection

Injection of intracutaneous/subcutaneous hydrogen gas

H2 gas (van der Waals force)

Due to the presence of intermolecular forces, it is not absorbed immediately

Diffusion to body fluid / interstitial fluid (molecular hydrogen): Transition part: 1.6 ppm? effect

Transition into the blood / lymph fluid: Transition part: 1.6 ppm effect

Subcutaneous emphysema condition

Hydrogen Gas injection

Gas concentration in cylinderFilled gas concentrationHumidityFilling pressureDose

: >99.999% : 99.9999% : 0% : 0~100kPa : Xml

· Cell/tissue damage due to filling pressure· Dosage by region

OH NFκB↓ COX2↓ Inflammation↓☓

TNFα/IL-1

Sterile H2 gas

Inflammation and reactive oxygen

TNFα

UV/Ra

LPS

CD40/RANKLT-β

IL-1

NFκB COX2 Inflammatory response

Nrf2

H2

NOX ↓ O2

-

↓ H2O2

Fe Cu Sterilization

Intra-articular injection

Hypothesis of pain relief

Aseptic 100% hydrogen gas administration into the joint space(Administration to closed space in joint)

↓Dissolution in synovial fluid/synovial fluidInvasion into the joint capsule

↓Reduction of inflammation/pain relief

Transdermal absorbent bath agent/external medicine

Percutaneous absorption external use/bath preparation

O2 gasH2

waterEpidermis

Dermis

Subcutaneous tissue

Muscle tissue

Blood

Hydrogen molecules dissolved/reserved in water pass through

In the gaseous state, by intermolecular force H2 do not break through the skin because they are bonded

H2 gasH2 gas

Dissolution treatment

Hydrogen gas dynamics in the body by bathingChange in hydrogen concentration in exhalation gas by bathing

(15)

10 20 30 40 5010

14

16

18

20

beforeafter

ppm

min

12control

Hydrogen bath agent

Medical Corporation Medical Association Medical Association Tsuji Clinic

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HyCare Akarui Ltd