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NADH in Cancer Prevention and Therapy

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Phytopharmaceuticals in Cancer Chemoprevention and Therapy 2

 

Phytopharmaceuticals in Cancer Chemoprevention and Therapy

Chapter 12: NADH in Cancer Prevention and Therapy

George D. Birkmayer, MD, PhD

Department of Medical Chemistry, University of Graz and Birkmayer Laboratories, Vienna, Austria

Jiren Zhang

Oncology Center, Zhujiang Hospital, Guangzhou, P.R. China

1. Biological functions of NADH
NADH is the abbreviation for Nicotinamide Adenine Dinucleotide Hydride. NADH is also known under a number of other synonyms such as: Nadine, disodium salt, reduced form Diphosphopyridine nucleotide, reduced form Adenine-D-ri bose-phosphate-phosphate D-ribose-nicotinamide, reduced form Cozymase, reduced form Coenzyme 1, reduced form Codehydrogenase, reduced form NADH is present in every living cell where it catalyzes more than a 1000 biochemical reactions. The most important biochemical functions of NADH are the following:

NADH is the cellular fuel for energy production
NADH plays a key role in DNA and cell damage repair
NADH enhances the cellular immune system
NADH is the most potent antioxidant
NADH stimulates dopamine and adrenaline biosynthesis
1.1  NADH is the fuel for cellular energy production
All living cells require energy to stay alive. Without energy, a cell dies because the energy production represents the essential prerequisites for every living cell (1).

How is energy produced in the cell? NADH reacts with oxygen to produce in a cascade of biochemical reactions water and energy. This energy is stored in form of the chemical compound adenosine triphosphate,  abbreviated ATP. NADH itself is produced from amino acids, sugars and lipids via the citric acid cycle. One molecule of NADH yields three molecules of ATP and the more NADH a cell has available, the more energy it can produce. The amount of NADH a cell contains depends on the amount of energy it requires. Heart muscle cells, which have to contract themselves every second for entire life-time 86400 times a day, contain 90 mcg of NADH per gram tissue. Brain and muscle cells contain 50 mcg. One third of all the energy produced by our body is used up by our brain.

NADH increases ATP production in heart cells
In a recent study it has been found that NADH can increase the biosynthesis of ATP inside the cell. Isolated single heart cells were incubated with NADH (NADH was outside the cell). An increase of ATP inside the cell was found by two independent methods (Pelzmann et al. 2003). This observation provides convincing evidence that NADH can increase the cellular energy level in form of ATP.

If the cell has more energy, it can live longer and can perform its functions better.

The consequences are as follows: Heart cells get more energy by NADH, hence their strength and capacity is higher. People with heart problems can benefit from NADH. After a heart attack some areas in the heart may be damaged and hence not functioning. However, these cells are not totally vital. By providing these cells NADH, they may get more energy to repair the damage and will become functioning again.

The same principle may work in the brain. After a stroke, certain areas in the brain are not nurtured by blood as the circulation is blocked. These brain parts are to a certain extent still vital but not functioning, however, not totally dead. By offering them NADH they get more energy and may regain their functionality. A number of anecdotal cases of stroke patients treated with NADH did show improvement of their symptoms even weeks after the event.

If NADH works in isolated heart cells it should also work in other tissues such as the kidney, the liver, the pancreas or the lung.

NADH has been shown to increase the mitochondrial membrane potential. Mitochondria are the power plants of the cell. There NADH reacts with oxygen to produce in a cascade of reactions energy in form of ATP

The site of this process is the membrane of the mitochondria, particularly the inner part of it. The British noble/ laureate Peter Mitchell postulated that energy in the mitochondria is formed by an electric charge between the outer and the inner side of the mitochondrial membrane. The higher level this electric potential reaches, the more energy is produced. Researchers in China could demonstrate that incubating cells with NADH leads to an increase in the mitochondrial membrane potential (Ref. Xu Meng) indicating more energy output.

NADH is taken up by blood cells.

The dogma stated in text books of biochemistry says NADH does not pass the cell membrane, hence cannot be provided from outside the cell to give it additional energy.

This dogma seems to be falling apart. Scientists have convincingly shown NADH can increase ATP formation and energy production in isolated cells (Pelzmann et al. 2003). In doing so, NADH has to penetrate the cell membrane to get to the point of action, the mitochondria. NADH is also taken up by cells lacking mitochondria such as erythrocytes. If you incubate human red blood cells with NADH a decline of the extracellular NADH and an increase in ATIP (= energy) in these cells is observed. The consumption of NADH by blood cells correlates (indirectly) to the level of ATP. In other words, if blood cells consume a lot of NADH, the ATP level in these  cells is low.

Highly conditioned athletes are assumed to have in their muscles and blood cells a respectively high energy level. Hence their blood cells can consume only a low amount of NADH when they are incubated with NADH. Blood cells from elderly or sick people consume considerably more NADH than athletes. However, when athletes are tested after a marathon run or a long distance cycling their blood cells consume about the same amount as those of old people. This observation has been shown with a new NADH consumption test (Ref. Nadlinger).

This new blood test called ENMA (Extracellular NADH Metabolization Assay) has an enormously broad application range. It can be used for controlling the training of athletes but also for the surveillance of patient’s energy recovery, particularly after heart-, stroke or cancer treatments.

NADH plays a key role in DNA and cell damage repair
The DNA is well protected in the nucleus by histones and other macromolecules. Nevertheless, it can be damaged by exposure to various agents such as radiation, UV light, ozone, carcinogens and toxins such as cytostatic drugs some of which are also carcinogenic. These potentially harmful agents can react with our chromosomes. If our DNA is affected and damaged by one of these agents our genetic material will be altered. Replication of altered, 6 defective DNA causes changed features in newly divided cells provided cell division can still occur. The greater the DNA damages the more extensive alterations in the cell and tissue occur (7). Genetic damage is the biochemical basis for a number of chronic diseases such as cancer (8,9), rheumatoid arthritis, immunodeficiences and arteriosclerosis (9,10). Hence it is imperative that our genetic material remains unaltered in order to guarantee that any new progenitor cells developing after cell division occurs are identical to their parent cells. If the DNA is altered by physical or chemical agents, the newly developing progenitor cells may be different from their mother cells and will not function in the programmed way.

In order to avoid the fatal consequences of DNA damage, mammalian cells have developed a system which is able to repair alterations to their genetic material. This so-called DNA repair system needs NADH to gain full functionality (11, 12). Therefore, the more NADH you have in your body, the better the DNA repair system functions and the better you are protected from potentially developing diseases.

(Arbeit Zhang) The exposure of cells to DNA-damaging reagents can trigger a wide range of cellular responses involved in the regulation of gene expression and cell-cycle progression, stimulation of DNA repair and programmed cell death (1-2). These progresses are important for maintaining normal growth, anti-mutation, damage repair and functional activity of cells. However, due to the unspecificity of chemotherapeutic drugs for the target cells, many normal cells in addition to the cancerous ones get damaged causing severe, sometimes fatal adverse reactions. The question is how can normal cells be protected from the cytotoxic effects of chemotherapeutic agents? How can we stimulate the repair system and promote normal cellular responses after chemotherapy? The mechanism involved in repairing DNA-damaged cells exposed to cytostatic has been investigated in many clinical studies. (1-5) Whether the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH) can be used to protect cells from DNA-damage has never been considered until recently. Previous studies in our laboratory have found that NADH can stimulate biosynthesis of endogenous cell factors, and can rescue cells from apoptotic damage by triggering production of the bcl-2 oncogene proteins (6).

The effect of NADH on DNA repair was investigated on PC1 2 cells, damaged by doxorubicin. PC12 cells were incubated in medium without and with NADH before and after exposure to the DNA damaging agent doxorubicin. The changes of the cell proliferation genes (c-myc, c-erbB-2) the apoptosis inhibition gene bcl-2 and p53 (tumor suppressor gene), cell apoptosis inhibition gene bcl-2 and p53 (tumor suppressor gene), cell apoptosis gene (c-fos) and the proliferating cell nuclear antigen (PCNA) were investigated using a cytotoxicity assay and immunofluorescence flow cytometric analysis.

Doxorubicin induced DNA damage in PC12 cells by inhibiting the expression of the cell proliferation genes and by triggering apoptotic processes in the cells. This was shown by down regulating the expression of c-erb-2, c-myc, bcl-2 and upregulating the expression of PCNA and c-fos of the PC1 2 cells.

NADH did not only increase the resistance of PC 12 cells to the doxorubicin induced DNA damage but also repaired the  damage  partially.  NADH  promoted  survival  and  differentiation by regulating the  c-myc  oncogenes protein.

Furthermore it supported the process of DNA repair by regulating the expression of p53, bcl-2 on the PC 12 cells damaged by doxorubicin. NADH also downregulated expression of the cell apoptosis gene c-fos on the PC12 cells.

The expression of c-ervB-2 oncogene protein and PCNA on the PC12 cells did not show a significant change in the group of cells incubated with NADH in comparison to the group incubated with medium alone. In addition, an abnormal proliferation effect of NADH on PC12 cells has not been observed in these experiments. As  a consequence of these findings, NADH may be considered as a therapeutic adjunct for cancer patients to protect them from toxic substances such as doxorubicin or cisplating by stimulating the DNA repair system and by promoting normal cellular biosynthetic responses after chemotherapy.

Drug-induced apoptosis is dependent on the balance between cell cycle checkpoints and DNA repairing mechanisms. Doxorubicin is a DNA-damaging cytotoxic drug, which is found to accumulate in the nuclei of damaged cells. Increased accumulation of cellular doxorubicin is accompanied by apoptosis (1,2,12). Experiments indicate that the inhibition rate of PC12 cells correlates with the concentration of doxorubicin in medium and with time of exposure of the cells to the toxic environment.

The cytotoxicity of doxorubicin for PC12 cells occurs not only in the phase of acute exposure but also in the lag phase.

Apoptosis induced by doxorubicin is accompanied by the down-regulation of the expression of the oncogenes proteins c-erb-2 and c-myc, the anti-apoptotic gene proteins (bcl-2), p53 tumor suppressor protein and upregulation of the expression of PCNA (3) and c-fos.

These genetic changes occur not only in the early phase of the apoptosis induced by doxorubicin, but can also happen in the lag phase, when the damaged PC12 cells are incubated with new medium after removing the old medium containing doxorubicin. DNA damage and activation of c-fos oncogene seem to be the major pathways of inducing apoptotic damage of PC12 cells. NADH can partially rescue cell activity of PC12 cells from DNA damage induced by doxorubicin. Cell damage repair is a complex biological process in which a number of reactions are involved.

NADH is an essential component of enzymes necessary for many metabolic reactions in the cell including energy production. It plays a crucial role in triggering biological antioxidation and in regulating the expression  of membrane glycoprotein receptors (20, 21). Previous studies have shown that NADH can rescue cells from apoptosis caused by inhibition of the mitochondrial respiratory chain induced by chemotherapeutic agents, and simultaneously can increase the production of endogenous biological factors necessary for proper functions. In addition, cell cycle progression of PC12 cells is observed (2,6).

NADH can repair DNA damage. The cytotoxicity test and flow cytometric assays indicate that the repair ability of NADH on a damaged cell depends on the degree of DNA-damage of PC12 cells.

When the apoptotic rate of PC12 cell was 82.2%, the rate of cells repaired by NADH was only 3.1 %. After recovery incubation for 48 hours, the expression of c-erbB-2 oncogene proteins and PCNA on the PC12 cells did not show a significant increase in the group treated with NADH in comparison to the control.

The significantly abnormal proliferation effect of NADH on PC12 cells has been observed. The change of cerbB-2 oncogene happening in the acute damage phase of the PC12 cells is difficult to be rescued by incubation with NADH or medium. However, the upregulation of cfos oncogenes protein in the acute damage phase can be significantly down-regulated by incubation with NADH for 48 hours. This suggests that NADH rescues PC12 from doxorubicin induced damage not only by repairing the DNA but also by increasing energy production in these cells.

Programmed cell death is an energy dependent biochemical regulated process that is the result of the expression of a number of genes. The roles of several gene and gene families such as Bcl-2/bax, P53, c-myc, c-jun,  c-fos, considered to be critical for apoptosis have recently been described in different cell lines (23,24). Many reports suggest that a rather complex genetic and molecular mechanism is involved in the process of apoptosis. It could also 10 be triggered either by increased or by reduced gene expressions as well as by biochemical reactions not necessarily connected to altered gene expression (1,22,23,24,25). Observations from our studies provide evidence that complex molecular events are involved in the apoptotic process of PC-12 cells induced by doxorubicin. After recovery incubation of PC-12 cells with NADH for 48 hours, the positive ratio and amount of c-erbB-2 expressed on PC12 cells did not show an increase in comparison to the control with medium alone group. The positive ratio of c- myc was not altered, but the amount of c-myc expressed on the vital PC12 cells was significantly upregulated 47.7% and 52.9% in comparison to the acute damage phase and the group with medium alone. This suggests that regulating the expression of c-myc on PC1 2 cells may be involved in the DNA repair of PC1 2 cells damaged by doxorubicin. Although the exact function of c-myc remains largely unknown, its activation has been implicated in the induction of cell proliferation and differentiation has been implicated in the induction of cell proliferation, and differentiation. Some reports have also indicated that the cmyc oncogenes protein acts as sequence-specific factor that serve to regulated gene expression in normal cellular growth and differentiation and as a common intracellular transducer which promote Go to G, transition. They may also be involved in the regulation of programmed cell death (27,28,29,30).

NADH repairs DNA-Damage of PC12 cells by regulation of p53 and bcl-2 gene protein expression.

In the processes of cell DNA damage and repair, bcl-2 and p53 are the two of the most important proteins encoded by the bcl-2 gene and p53 tumor suppressor gene. Wild-type p53 can suppress cell proliferation and slow DNA synthesis and block transition from G1 to S phase of the cell cycle (30,31,32). Bcl-2 is a proto-oncogene and the most important inhibitor of apoptosis. Expression of bcl-2 may interfere with the apoptotic process mediated by the APO-1/Fas antigen and TNF receptor. Probably the ration of bcl-2 and p53 determines how the cell responds to DNA-damaging agents. Current research indicates that expression of bcl-2 in Pheochromocytoma cells is associated with that of the c-myc oncogene protein (34,35,36). Overexpression of the proto-oncogene bcl-2 might block p53- induced apoptosis and inhibit p53 functional activity (37). In our experiment, in which we investigated the effect of NADH on the recovery of PC12 cell from DNA-damage, the ration of expression of p53 and bcl-2 on PC12 cells was down-regulated by 91.9% and 98.8% by exposure of the cells to doxorubicin in medium. After recovery incubation in medium with NADH for 48 hours, the ratio of vital PC12 cells was upregulated by 3.1% and p53 tumor suppressor protein expressed on the vital cells down-regulated 36.7%. However, the amount of bcl-2 expressed on the vital PC12 cells was found to be upregulated by 12.7% in comparison to the control  group (medium alone). These findings suggest that NADH can not only promote survival and differentiation by regulating the c-myc oncogenes protein, but also support the process of DNA repair by regulating the expression of p53 tumor suppressor protein and protooncogene protein bcl-2 on the PC12 cells damaged by doxorubicin.

Many so-called cytostatic drugs, such as cisplatin and doxorubicin cause DNA damage and may thus be cancer causing agents. Nevertheless they are frequently used for treatment of cancer. This appears to be a paradoxon that cancer causing chemicals are used to treat cancer patients.

NADH has been shown to protect cells from damage by doxorubicin (Ref. Zhang, 98).

Cisplatin is one of the most frequently used drugs for chemotherapy of cancer. It damages the cell membrane mitochondria and the nucleus not only from cancer cells but from all (normal non cancerous) cells as well. The consequences are the so-called side effects of chemotherapy such as hair loss, gastrointestinal problems (vomiting, dizziness etc.)

Preincubation of cells which have been damaged by cisplatin with NADH prevents the changes induced by cisplatin (Ref. Meng Xu, 2000). Based on these findings, cancer patients should protect themselves by taking NADH when receiving Cisplatin, doxorubicin or other cell damaging cytostatic.

NADH stimulates cellular immunfaction
1.3.1 NADH stimulates the production of cytokines particular Interleukin-6

The activities of white blood cells such as the T-lymphocytes, the B-lymphocytes and the macrophages are triggered by ##########.

NADH is also involved in transcriptional pathways important for development, cell cycle regulation and transformation.

The corepressor CtBP (carboxy-terminal binding protein) binding to cellular and viral transcriptional repressors is regulated by the nicotinamide adenine dinucleotides NAD and NADH, with NADH being two to three orders of magnitude more effective (Zhang et al., Science 295, 1895-1897, 2002, Publ. 1.2).

The best-characterized target promoter for CtBP in mammalian cells is probably the Ecadherin gene (Grooteclaes et al., 2000, Rev. 16 of Science 295, u. Ref. 20 ibid.) Loss of Ecadherin expression in turnours correlates with metastasis, invasion and poor clinical prognosis (Ref. 21 and 22 ibid.).

It has been shown that CtBP-mediated repression of the E-cadherin promoter is enhanced by hypoxia (Zhang et al. wie oben 1.2) NADH may alleviate the hypoxia by stimulating oxygen uptale into the cell. It has been shown that the oxygen uptake in the muscle of highly conditioned athletes increases when they were taking the stabilized orally absorbable form of NADH (Birkmayer, 2003, ICMAN publ). NADH has been shown to be a sensor of blood flow needed in brain muscle and other tissues (Ido et al, FASEB 15, 2001). Increasing blood flow removes lactate and augments delivery of fuels and oxygen for energy metabolism.

NADH stimulates cellular immune functions
The cellular immune response in humans is based on the activities of the T-lymphocytes, the B-lymphocytes and the macrophages. Macrophages have the capability for direct elimination of allogen entities such as bacteria and other foreign tissues. The first step in the elimination of bacteria is the perturbation of the plasma membrane of the macrophages. As a consequence the metabolic activity including oxygen consumption is marked increased. Most of oxygen is converted to superoxide and hydrogen peroxide (#31#Ref. 13, NADH Bochlein). This phenomenon, known as “metabolic burst” appears to be the first and most critical step leading to the destruction of the invading foreign organism. During this metabolic burst and in the cytotoxic activity of the macrophages high amounts of NADH are used. Hence the immune defense mechanism of white blood cells is fuelled by NADH. Furthermore it has been shown that NADH stimulates the biosynthesis of interleukin-6 (IL-6). Peripheral human blood leucocytes when incubated with NADH significantly stimulate the release of IL-6 a dosage dependent manner. (#32#Nadlinger et al., 2001 siehe Publ. 1.3).

IL-6 has been reported to protect neurons from degeneration the mechanism of which has not yet been elucidated. If IL-6 protects neurons it may protect other cells as well.

NADH is the most powerful antioxidant
An antioxidant is a substance which acts against oxidation. The opposite of oxidation is reduction. Compounds with a high reduction potential exhibit a strong antioxidant power. NADH, the reduced form of Coenzyme 1 has the highest reducing power as a single molecule of any biological material. Only molecular hydrogen has a higher reduction potential but does not exist in living cells. Biological antioxidants are present in all living cells to protect the cell and its membrane from destruction by free radicals (#33#Ref. 15, NaDH Bachl.). Free radicals are molecules with an unpaired electron. Hence they are extremely reactive. They interact with many compounds in human cells, in particular with the lipid-containing structures such as the cell membrane. In doing this, they violate the integrity of the cell wall causing leakage and release of essential cellular components which usually results in cell death (Ref. 16, NADH Buch). Free radicals have been shown to be involved in the development of cancer (#34#Ref. 17, NADH Buch) coronary heart disease atherosclerosis, diabetes, neurodegenerative disorders and autoimmune diseases (#35,36#Ref. 18, 19, NADH Buch). Free radicals are formed in human cells by agents knocking out electrons from a molecule. These agents can be x-rays or other forms of high-energy radiation such as the one used for radiotherapy of cancer. Small amounts of free radicals are also produced in normal cells by metabolic reactions. However, mammalian cells possess a defense system to protect them from being irreversibly damaged (#37#Ref. 21, NADH Buch). This system is called “antioxidative protection shield”. The first and most important antioxidant component in this system is NADH, because it has the highest reduction potential of any compound in the cells (#38#Ref. 22, NADH Buch). A measure for free radical formation and lipid peroxidation concomitantly are the thiobarbituric acid reactive species (TBARS) in a study using spontaneous hypertensive rats (SHR). It has been shown that the renal TBARS were significantly lower (1.9nmol/MAD/100 mg tissue) in the rats red with 5 mg NADH  orally as compared to the control animals (3.5 nmol/MDA/100 mg tissue). IVIDA is the abbreviation for malondialdehyde which is formed from the breakdown of polyunsaturated fatty acids. NADH also reduced total cholesterol and ILDL-cholesterol significantly as well as the blood pressure (#39#Busheri et aL, 1998). One of the conclusions   the authors of these papers made was NADH may turn out to be a useful agent to prevent and treat cardiovascular risk factors common in aging. The antioxidative effect of NADH was also investigated in humans. When ILDIL cholesterol is oxidized in vitro induced by peroxyl radicals NADH reveals an antioxidant effect identical to ascorbic acid during the first 90 minutes. However, after 90 min ascorbic acid has no further effect while NADH  is still acting antioxidatively after 90 minutes. Hence the antioxidative potency of NADH is much longer lasting than that of ascorbic acid. In a double-blind placebo controlled study 37 human subjects were either taking ENADA-NADH (4 x 5 mg NADH) tablets or placebo tablets for 4 weeks. NADH caused a reduction of malondialdehyde as well as in the oxidative stress-induced carbonyl modification of proteins particularly in smokers. A steady decrease of the initially elevated protein carbonyl modification levels of smokers was observed which approached the levels of non- smokers within the study period of 4 weeks. This observation implies that ENADA-NADH has some kind of curing effect on the alterations and damage caused by cigarette smoking.

ENADA – the stabilized orally absorbable from of NADH
NADH can be regarded as biological form of hydrogen. Hence it is a very reactive compound. Due to this NADH is rather unstable and gets easily degraded by air, water, humidity, acids and oxidizing agents such as sugars. Even in solid state NADH reacts with lactose, the most common filler of tablets. In 1987, NADH has been used intravenously for treatment of Parkinsonian patients. The beneficial effects in improving the disability of the PID patients were remarkably (#40# Birkmayer et al., 1988). The challenge was to transpose the im. form into an oral (tablet) form. After yearlong research a galenic formulation was developed in which NADH was stable for at least 2 years a prerequisite for registration as an ethical drug. For the formulation of a stabilized orally absorbable from one of the authors (G.B.) received worldwide patents (ref. Pat. USA, EU, Japan, China, Ref. 41 NADH Buch#41#). The brand name for the patented stabilized orally absorbable form of NADH is ENADA. Numerous studies have been performed since the development of ENADA in 1993.

Bioavailability of ENADA – NADH
The stabilized form of NADH when taken orally is absorbed in the small intestine. Studies have shown that NADH passes the intestinal mucosa by passive diffusion (Ref. Mattern Diss.)

When rats were fed with one tablet ENADA-NADH 5 mg an increase of the NADH level in the brain cortex after 20 minutes of intake could be measured by laser-induced fluorescence (Rex et al.).

In another study the bioavailability of NADH in the central nervous system was measured by Laser-induced fluorescence spectroscopy. Using a pulsed N-2 laser combined with a fibre optic probe and photomultipliers the NADH fluorescence was measured in the brain cortex of rats. After intraperitoneal application of NADH (50 mg/kg) an increase in the intensity of the cortical NADH fluorescence of about 18 % was observed for approximately 30 minutes compared to the fluorescence intensity in the control group. Neither NAID+ (the oxidized form of NADH) at concentration of 50 mg/kg nor nicotinamide (50mg/kg) did show any effect on the NADH fluorescence in the cortex for the entire measurement of 120 minutes.

Following oral application of NADH (2 tablets of ENADA 5mg NADH = 51 mg/kg) the cortical fluorescence intensity was increased by about 20% compared to the control group. (Rex et al. Pharm & Tox 90, 2002). The results of this study provide evidence that NADH given orally increases the amount of NADH in the brain. These findings imply that the stabilized orally absorbable form of NADH (ENADA) passes the blood brain and can reach the brain where it can evoke its action.

ENADA – NADH – a protector against chemotoxicitV and
Cytostatic drugs such as Cisplatin and Doxorubicin are used in the chemotherapy of cancer. These drugs trigger a wide range of cellular responses involved in the regulation of gene expression and cell cycle progression and programmed cell death.

As these cytostatic drugs are not specific cancerous as well as normal cells get damaged causing severe sometimes fatal adverse reactions. Studies from the authors have shown that NADH can stimulate the biosynthesis of endogenous cell factors and can rescue cells from apoptotic damage by triggering production of the bcl-2  oncogene proteins (Zhang et al. J.Tum Marker Oncology 13, 11-24 (1998). In the process of cell damage repair bcl-2 and p53 are the two of the most important proteins encoded by the bcl-2 gene and p53 tumor suppressor gene. Wild type p53 can suppress cell proliferation and can slow DANN synthesis and thus block transition from G1 to S phase of the cell cycle. (Blagosklonny et al. Ref. 31 von Zhang paper). BcI-2 is a proto-oncogene and the most important inhibitor of apoptosis. Current research indicates that expression of bcl-2 is associated with that of the c-myc oncogen protein (Ref 34.Zhang Doxo paper). IN studies performed by the authors it was found that Doxorubicin down regulated the expression of p53 and bcl-2 in PC 12 cells by 91.9% and 98.8% respectively. Incubation of the damaged cells by NADH promoted survival and differentiation by regulating the c-myc oncogene protein. NADH also supports the process of DANN repair by regulating the expression of the p53 tumor suppressor protein and the protooncogene protein bcl-2. (Zhang et al. J.Tumor Onc. 13, 1998). Similar results were obtained when Cisplatin was used as cell-damaging agent.

Further studies investigated the effect of NADH on cells damaged by radiation.

PC12 cells (???) were damaged by radiation in the dose used for radiotherapy of cancer. Radiation caused a 90 % (???) damage of vital cells Incubation of the damaged cells with NADH induced a repair process. Of the 90% damaged cells 70 % (???) could be repaired and gained full functionality. (Zhang et al. siehe ICIVIAN Vortrag).

A number of cytostatic drugs including Cisplatin are carcinogen and can cause cancer by themselves. NADH is able to protect cells from the carcinogenic effects of these chemotherapeutic agents. NADH can serve as protector against at least against certain carcinogens. Hence ENADA, the stabilized oral form of NADH, may present a safe, nontoxic biological tool for prevention of cancer.

The safety of ENADA-NADH
The stabilized orally absorbable form of NADH (ENADA) is a nutritional supplement available in the U.S. since 1995 and in the E.U. since 1997. Based on the patented formulation of ENADA a number of clinical trials have been launched to prove scientifically that ENADA is effective. In order these studies could get started an Investigational New Drug Application (IND) was filed with the Food and Drug Administration (FDA). To get approval from the FDA for these studies it has to be proved that ENADA (the stabilized oral form of NADH) is safe. Before testing the oral form of NADH the maximum tolerated intravenous dose (MTD) of RNADH (reduced form of beta- nicotinamide adenine dinucleotide) in beagle dogs was elucidated. It was found that the maximum tolerated dose (MTD) of RNADH in dogs is 500 mg NADH per kg per day. In other words a 10 kg heavy dog will tolerated 5 grams of NADH. In addition the oral form of NADH (ENADA) was tested in beagle dogs. 150 mg/kg/day were given form 14 days. This corresponded to 30 regular (5 mg) ENADA tablets filled in 2 gelatine capsules (15 ENADA tablets per capsule). This high dose was selected because it was considered to be the maximum amount which could be practically administered repeatedly over 14 days. All dogs survived the treatment and no adverse reactions or side effects have been observed.

The dogs treated with ENADA showed no changes in comparison to the control animals regarding laboratory safety parameter and organ and tissue pathology.

150 mg /kg bodyweight means 1500 mg for a 10 kg beagle dog. 1500 mg of ENADA correspond to 300 ENADA 5mg tablets per day. This is a dose which beagle dogs tolerate without any side-effect. (Birkmayer et al. 2003 manuscript submitted)

In addition to the MTD studies a study for chronic toxicity was performed in rats.

Rats were given 1 tablets ENADA (5mg NADH) per day for 26 weeks. No changes in laboratory parameter and in tissue and organ pathology were observed. (Birkmayer & Nadlinger 2002). 5 mg for a rat weighing about 330 grams corresponds to 15 mg per kg bodyweight or 1050 mg of NADH for a 70 kg heavy human subject. 1050 mg NADH correspond to 210 tablets of ENADA (5mg NADH) which are tolerated without side effects when given for 26 weeks (6.5 months). Based on these safety data ENADA-NADH can be generally recognized as safe.

ENADA-NADH as therapeutic concept for certain human cancers
Based on the various biological functions ENADA the stabilized oral form of NADH has been used for treatment of cancer patients.

A number of anecdotal cases will be described in the following in which ENADA-NADH was used as anti-cancer therapeutic approach.

Case 1: 48 year old male suffering from a small cell bronchial carcinoma. The diagnosis was made by IVIRT (magnetic resonance tomography) and verified by histopathological examination of biopsy specimen. The size of the tumor was 8 to 10 cm in diameter (about the size of a tennis ball) in Sept. 2001 when the patient visited one of the author (G.B.) The report of the university of Amsterdam indicated the tumor was inoperable due to its localization very closed to the mediastinum and partially penetrating it. The patient were recommended to take 4 tablets ENADA 5mg NADH per day. He was already taking Selenium, vitamin C and vitamin E.

In January the size of the tumor was verified by IVIRT to be the size of a cherry.

The therapy with NADH was continued. In July 2002 a IVIRT report of the university Of Amsterdam indicated the tumor has disappeared.

For the time being we can only speculate on the mechanism of action of NADH in curing patients from cancer.

One possibility could be the role of NADH in DNA repair. Cancer cells have a DANN which is altered in comparison to the original cells from which the carcinoma cells did develop. If NADH is given to cancer patients the content of NADH in the cancerous cell increases. The more NADH a cell has available the better the DNA repair system works and the alteration of the genes may be reverted to normal. Another possibility may be derived from the energy increasing function of NADH. It has been shown that the intracellular level of ATP can be increased by incubating the cells with NADH (Pelzmann et al. Br.J.Pharm. submitted 2003). With more energy cells increase their capacity of the biosynthesis of macromolecules in particular proteins, glycoproteins and glycolipids. These substances play a major role on the cell surface in regulating proliferation and differentiation. With more NADH and ATP in the cancerous cells proliferation may be halted and differentiation processes may be induced. (Ref.??). These assumptions remain to be elucidated in further studies.

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