Gene-Eden-VIR Decreased Physical and Mental Fatigue in a Post Marketing Clinical Study That Followed FDA Guidelines; Results Support Microcompetition Theory ()
1. Introduction
This paper reports the results of a post marketing clinical study that tested the effect of Gene-Eden-VIR, an antiviral natural formulation, on fatigue. Specifically, the clinical study tested the effect of Gene-Eden-VIR on the feeling of general, physical, and mental fatigue. The population included individuals infected with a latent virus, including the Human Papillomavirus (HPV), Epstein Barr Virus (EBV), Herpes Simplex Virus (HSV), Human Cytomegalovirus (HCMV), and Hepatitis C Virus (HCV).
Studies have shown that one-third of the general population reports a feeling of fatigue, with 5% reporting a feeling of short-term fatigue (<6 months), and 31% reporting a feeling of long term or chronic fatigue (>6 months) [1] . In the US alone, the feeling of fatigue is responsible for 10 - 15 million visits to the family doctor every year [2] .
Studies associate the feeling of fatigue with a sense of tiredness, lack of energy, a feeling of exhaustion, and impaired physical and/or cognitive performance [3] .
Various diseases (e.g., anemia, multiple sclerosis, CFS), psychiatric disorders, and drugs (e.g., antihypertensives, β-adrenergic blockers, lipid-lowering agents) can cause a feeling of fatigue. One of the most important causes of a feeling of fatigue is an infection, specifically, with a latent virus, such as the Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis viruses, parvovirus B19, and influenza virus [4] -[7] .
Most scientists and physicians believe that latent viruses cause no pathologies or symptoms. The FDA even states on its website: “Some viruses, however, can enter a state known as latency in which the virus is not being replicated. In the latent state, the virus does not cause disease” [8] . As a result, current standard medical practices don’t target latent viruses.
In contrast to this belief, the Microcompetition with Foreign DNA theory [9] , proposed by Hanan Polansky in 2003, describes how viruses, during their latent phase, can cause a chronic disease.
The theory was presented in a book entitled “Microcompetition with Foreign DNA and the Origin of Chronic Disease”. The theory describes the effect of Foreign DNA fragments that include the cis-regulatory element, called an N-box, on transcription of cellular genes.
Consider foreign N-boxes that entered the body naturally, through, for instance, an infection or digestion, or artificially, through, for instance, an injection of some treatment. The foreign N-boxes attract the transcription complex GABP∙p300. Since the complex is a scarce genetic resource, the foreign N-boxes decrease the availability of this complex to cellular genes. As a result, the genes that are transactivated by the GABP∙p300 complex produce fewer proteins, and the genes that are suppressed by this complex produce more proteins. The abnormal levels of these cellular proteins cause a disease.
It is interesting that many common viruses that establish a latent infection have strong N-boxes in their promoter/enhancer. They include the Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Herpes Simplex virus (HSV), Varicella Zoster virus (VZV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), and the Human Papillomavirus (HPV). In fact, the CMV has the strongest promoter/enhancer known to science. Liu et al. showed that the CMV promoter/enhancer, which includes the N-box, is more than 150-fold stronger than the promoter of the platelet-derived growth factor-b chain (PDGF-b) gene [10] .
During latency, an infected cell harbors about 10 copies of the CMV [11] . Multiplying the two numbers, about 10 copies of CMV per cell during latency, and 150-fold stronger promoter/enhancer, suggests that a latent infection with CMV has a similar effect on the PDGF-b promoter, and hence, its transcription, as the introduction of 10 × 150, or 1500 copies of additional PDGF-b genes into the cell. Since, PDGF-b is susceptible to microcompetition with CMV [12] , the Microcompetition theory predicts that a latent infection with CMV will cause a decrease in PDGF-b transcription, a decrease in the concentration of the PDGF-b protein, and disease.
Studies have shown that GABP is involved in the mitochondria electron transport chain. For instance, several studies have shown that GABP transactivates the expression of the gene encoding the cytochrome oxidase subunit IV (COXIV), a terminal enzyme of the mitochondrial respiratory chain [13] [14] . GABP also transactivates the COX subunit Vb gene, another enzyme of the mitochondrial respiratory chain. Several studies indicated a direct role for GABP in the expression of all 10 nucleus-encoded cytochrome oxidase subunits. For instance, Ongwijitwat et al. associated GABP in vivo with multiple COX subunit promoters by chromatin immunoprecipitation [15] . Ongwijitwat et al. also showed that expression of a dominant negative GABP allele decreased COX expression, and that an siRNA directed against GABPα decreased expression of all 10 COX subunits [16] . In addition, studies identified functional N-Boxes in a number of other genes related to the respiratory chain. These include genes for Tfam [17] [18] and TFB1M and TFB2M [19] [20] , and three human succinate dehydrogenase (complex II) subunit genes [21] -[23] .
Other studies have also shown that GABP transactivates the expression of the neuromuscular proteins utrophin [24] , acetylcholine esterase (AChE) [25] , and acetylcholine receptor subunits delta (AChRd and epsilon (AChRε) through their N-box promoter/enhancer cis-regulatory element [26] .
Tinsely et al. showed that a small molecule that upregulates utrophin expression decreased the dystrophic symptoms in the mdx mouse [27] . Specifically, the study showed that “Treatment results in reduced pathology, better muscle physiology leading to an increase in overall strength, and an ability to resist fatigue after forced exercise”.
Faber et al. showed that mutations in AChRe subunit gene are underlying the Congenital Myasthenic Syndromes (CMS) in nine patients of Dutch origin [28] . CMS are neuromuscular diseases that show abnormal muscle weakness and fatigue. It is interesting that CMS patients also show a marked deficiency in utrophin at the neuromuscular junctions [29] .
Acetylcholinesterase inhibitors (“anticholinesterase”) cause muscle spasms and twitches and decrease muscle strength. Payne et al. showed that administration of neostigmine, an AChE inhibitor, to an anaesthetized man can result in muscle weakness [30] . Caldwell calls it paradoxical weakness [31] since cholinesterase antagonists are known to increase muscle twitch tension [32] , and are used in reversing the effect of muscle relaxant administered during anesthetics. Stum et al. showed that AChE deficiency in a mouse model might be contributing to muscle stiffness and fatigability [33] . These studies suggest that an AChE deficiency might increase muscle stiffness and fatigue.
Gene-Eden-VIR was designed to target latent viruses in infected individuals. Recently, a clinical study, conducted at the Center for the Biology of Chronic Disease (CBCD), showed that Gene-Eden VIR is antiviral [34] . Since GABP∙p300 is a transactivator of several respiratory chain genes, the Microcompetition with Foreign DNA theory predicts that Gene-Eden-VIR will increase the production of cellular energy, and the overall level of energy available to the infected host. Also, since GABP∙p300 is a transactivator of several neuromuscular genes, the theory predicts that Gene-Eden-VIR will decrease muscle stiffness and muscle fatigue in the infected host.
This study was designed to test these predictions.
2. Methods
2.1. Ethics Statement
An informed consent was obtained from all subjects prior to conducting the phone interviews.
2.2. Objective and Framework
We used this post marketing study to test the efficacy, safety, and optimal use of Gene-Eden-VIR on individuals suffering from fatigue. Specifically, we tested the effect of Gene-Eden-VIR on the feeling of general, physical, and mental fatigue. The study followed FDA guidelines.
2.3. Treatment
Gene-Eden-VIR is a dietary supplement. A capsule of Gene-Eden-VIR includes 100 mg of quercetin, 150 mg of green tea extract, 50 mg of cinnamon extract, 25 mg of licorice extract, and 100 mcg of selenium. The treatment included 1, 2, 3, or 4 capsules per day, and lasted 2 to 54 weeks.
2.4. Questionnaire
As of today, there is no objective tool to measure fatigue. Currently, researchers and clinician assess fatigue assessments by using a number of subjective scales. We discovered that none of these tools are relevant for our research. Therefore, we used a self-developed questionnaire called the Fatigue Natural Origin Treatment Clinical Questionnaire (F-NotCiq). The F-NotCiq questionnaire is a patient reported outcome (PRO) instrument. We successfully used a version of this tool in our previous studies. The purpose of a PRO instrument is to capture the patient’s experience. In our current study, our main endpoint was the feeling of fatigue. Meaning, the objective of the study was to measure the effect of treatment with Gene-Eden-VIR on the feeling of fatigue as reported by the treated participants.
The study collected the answers to the F-NotCiq instrument by phone interviews. We used two independent companies that specialized in outbound call services for performing the interviewers, one company from the US and one from Israel. The interviewers were blinded to the objective of the study. All interviews were recorded.
2.5. Population
The study randomly selected participants from the Gene-Eden VIR customer database that includes all GeneEden-VIR current and past customers. The traditional response rate to phone interviews is 10% - 15%. Since the study was aiming to collect 100 - 150 interviews, the study used a computerized system to randomly create a call list of 1000 customers. The final list of participants consisted of 100 Americans, both male and female, ages 20 to 66, infected with the latent HPV, EBV, HSV, HCMV, and HCV.
Since the objective of the study was to test the effect of Gene-Eden-VIR on the feeling of fatigue, the study excluded participants who reported no feeling of general, physical, or mental fatigue. That is, it excluded participants who reported a 7 point score on the pre-treatment question in these sections. Such score indicates that the participant does not suffer from the specific aspect of fatigue indicated by the question regardless of the treatment the participant actually received. This exclusion still preserved the intention to treat (ITT) principle.
At the start of the study, all participants reported a feeling of fatigue. Specifically, 98 reported a feeling of general fatigue, 90 reported a feeling of physical fatigue, and 79 reported a feeling of mental fatigue.
The study considered participants, who stopped taking Gene-Eden-VIR for a month or more before data collection, as past users. All other participants were considered as present users.
2.6. Controls
The Gene-Eden-VIR post marketing study includes a pre-treatment concurrent control, and an historical control. To create an historical control, we divided the original test group into two subgroups, present users and past users. Generally, an historical control is a separate group. However, since we did not have a separate group of nonusers, we used the past users as a proxy for an historical control.
2.7. Statistical Analysis
We tested the statistical difference between the score of “pre-treatment”, which is the numeric answer each participant used to describe his level of fatigue before the treatment started, to the score of “post-treatment”, which is the numeric answer each participant used to describe his level of fatigue after the treatment was completed. We also calculated the delta (Δ), that is, the difference in scores between the answers to the pre-treatment and post-treatment question. Then, we tested the statistical difference between the deltas. These tests were performed in a then-test model for both present and past users.
Statistical analysis was performed using a two-tail t-test assuming unequal variances.
The research defined the primary endpoint as a statistically significant increase in the score from pre-treatment to post-treatment on the raw answers and on the deltas.
3. Results
The participants reported no side effects following treatment with Gene-Eden-VIR.
Following treatment with Gene-Eden-VIR, 72 out of the 98 participants (73.47%), who reported a feeling of general fatigue, reported a decrease in their fatigue, 56 out of the 90 (62.22%), who reported a feeling of physical fatigue, reported a decrease in their fatigue, and 36 out of the 79 (47.36%), who reported a feeling of mental fatigue, reported a decrease in their fatigue. Moreover, out of the 73 (73%) participants, who reported a feeling of both physical and mental fatigue, 62 (84.93%), reported a decrease in at least one of those types of fatigue (Table 1).
Following treatment with Gene-Eden-VIR, the participants reported a statistically significant decrease in every aspect of fatigue tested in the study (Table 2).
We could not test for a dose effect since the number of participants who took 1, 3 or 4 capsules per day was too small for statistical analysis.
To test for a duration effect, we compared the change (Δ) from pre-treatment to post-treatment in participants who took Gene-Eden-VIR for less then two months and those who took Gene-Eden-VIR for two months or more. The results showed that participants who took Gene-Eden-VIR for the longer period reported a larger decrease in their feeling of fatigue. Specifically, the participants reported a statistically significant decrease in their feeling of general fatigue, and in their feeling of both physical and mental fatigue (Table 3).
*N is number of Participants, **Pre-T is Pre Treatment, ***Post-T is Post Treatment. gen. is general, phys. is physical, ment. is mental.
Table 2. Aspects of fatigue, pre-treatment vs. post-treatment.
The statistical analysis was conducted using one score per participant. #N is Number of Participants, Pre-T is Pre Treatment, Post-T is Post Treatment.
*Δ is change from Pre-T to Post-T. Statistical analysis was performed using the change in scores from Pre-T to Post-T reported by present users only. The analysis used one score per participant equal to the average of the answers to questions F1 - F3 for general, and questions P1 - P6 and M1 - M4 for physical and mental.
To test for a possible interviewer bias, we compared the change (Δ) from pre-treatment to post-treatment collected by the American and the Israeli call centers. The change was tested for both feeling of general fatigue, and for feeling of physical or mental fatigue. In both cases, the difference between the answers was statistically not significant (p = 0.79, n = 254, for general, and p = 0.49, n = 582, for physical or mental, Table 4). This means that although the centers included different interviewers from different cultures working at different times of day, Americans working during the day and Israelis working during the night, the answers were similar. Hence, the results showed no interviewer bias (Table 4).
To test for a possible selection bias, we compared the change (Δ) from pre-treatment to post-treatment by the past and present users of Gene-Eden-VIR. The change was tested for both feeling of general fatigue, and for feeling of physical or mental fatigue. In both cases, the difference between the answers was statistically insignificant (p = 0.70, n = 247, for general, p = 0.15, n = 568, for physical or mental, Table 6). This means that, statistically, the answers by the present users are the same as the answers by the past users, and therefore, there was no selection bias (Table 5).
An issue unique to natural products is the concern about the therapeutic consistency of marketed products. Please refer to the discussion on this issue in the FDA guidelines for botanical New Drug Applications (NDA) [35] . To test the therapeutic consistency of Gene-Eden-VIR, we compared the two batches used by the participants. The capsules in these batches were produced at two different manufacturing sites, and completed about 10 months apart. The results showed that the answers given by the participants who used the capsules from Batch 1 were the same as those given by the participants who used the capsules from Batch 2 (p = 0.62, n = 256, for general, p = 0.66, n = 574, for physical or mental, Table 6). Hence, the results indicated that, although GeneEden-VIR is a natural product, its formula has therapeutic consistency (Table 6).
4. Discussion
This post marketing clinical study showed that treatment with Gene-Eden-VIR safely decreased the feeling of
Table 4. USA vs. Israel call centers.
*Data may include up to three answers per participant. **Data may include up to ten answers per participant.
*Data may include up to three answers per participant. **Data may include up to ten answers per participant.
*Data may include up to three answers per participant. **Data may include up to ten answers per participant.
fatigue in individuals infected with a latent virus. These results support the Microcompetition with Foreign DNA theory.
The results are consistent. We observed a statistically significant decrease in the feeling of general, physical, and mental fatigue.
The results also showed a duration effect. Participants treated for two months or more reported a larger decrease in their feeling of fatigue compared to those treated for less then two months.
The results are robust. They showed no interviewer bias, no selection bias, and therapeutic consistency of the Gene-Eden-VIR formula under varying manufacturing conditions.
The results in our study are consistent with the results reported in another study that tested the effect of an antiviral drug on fatigue. In this study, Watt et al. 2012 showed that treatment with the antiviral drug Valganciclovir improved physical and cognitive performance in patients suffering from fatigue. The study also showed that longer treatments were correlated with better outcomes [36] .
This post marketing clinical study does not include a placebo control, that is, it is not a double-blinded study. Placebo controlled studies are the gold standard in medical research in pre-marketing clinical studies. However, except in rare cases, post marketing studies do not use placebo controls. They use other controls recommended by the FDA.
The FDA guidance lists five types of controls for both pre-marketing and post marketing studies: 1) Placebo Concurrent Control, 2) Pre-treatment Concurrent Control, 3) Dose-response Concurrent Control, 4) Active (Positive) Concurrent Control, 5) External Control (Including Historical Control). The External Control “can be a group of patients treated at an earlier time (historical control)” [37] .
The Gene-Eden-VIR post marketing study is a change from baseline study that includes a pre-treatment concurrent control and a proxy for an historical control.
The results are not likely to be a placebo effect. The current predominant and well proven theories on the placebo effect suggest that its main mechanisms are conditioned reflexes and patient expectations [38] . The GeneEden-VIR product literature does not mention the possibility of a change in the feeling of fatigue. Hence, the participants in this study could not have been primed for, or expect the reported effects. This lack of conditioned reflexes and patient expectations minimizes the possibility of a placebo effect, and supports the possibility of a physiological effect.
All participants who started the study completed it; therefore, the study has no follow-up bias. It should be noted that although we tested for some biases, others are still possible, for instance, the non-responsive bias.
This study relies on patient reported outcomes (PROs). Past studies showed that PROs had a significant role in the development and evaluation of new medicines [39] . According to the FDA, PROs are a valid and valuable source for measuring the efficacy of new drugs. They are reliable enough to warrant an approval of a label claim for a new drug. From the years 1997 to 2002, the FDA approved 23 new drugs based on results obtained in studies that used only PRO endpoints. They include six anti-migraine products (Amerge®, Ax-ert®), several anti-epileptics (Gabitril®, Keppra®), and a variety of other therapy classes (Tamiflu®, Relenza®).
The FDA regards PROs as a valid and valuable source of data. The scientific community also believes that PROs are valid and useful. Many major journals published clinical studies that use patient reported outcomes. The trust of the FDA and the scientific community in PROs should convince the medical community, and specifically, doctors, to trust studies that use PROs when evaluating the benefits of new treatments.
A possible criticism of the F-NotCiq is that it uses a subjective scale, and therefore, may be producing biased results. However, the feeling of fatigue is essentially a subjective experience, and therefore, cannot be measured objectively.
The size of the study group is a major concern in clinical studies. A small group may fail to show a positive effect. In addition, a small group might misrepresent the diversity in the population. The standard principle for multivariate behavioral research is at least 10 patients for one endpoint, that is, for one dependent variable [40] . This study included one endpoint, the change in the feeling of fatigue from pre-treatment to post-treatment. The population included 100 individuals. Hence, the size of the study group is adequate.
One might also question the reliability of the participants recall due to the long duration of the period under investigation (up to 54 weeks). This study used a “then-test” method. This method, which is also known as the retrospective pre-test-post-test design method, asks participants at the post-test period to think back to the pre-test period, and retrospectively rate their condition. The “response shift” is defined as the difference between the “pre-test” and the “then-test” ratings. Currently, the response shift is a well documented and extensively researched phenomenon [41] . According to the literature on “response shifts”, participants may alter their internal standards, values, or conceptualization of their quality of life when experiencing changes in health states. The response shift can affect or distort the reported scores and undermine the credibility of the observed medical or psychosocial effects. Many studies reported that, after participants experience an improvement in their health, a then-test tends to show a decrease in the initial assessment of the original level of well being.
Since this clinical study uses the “then-test” method, we tested for a possible response shift by comparing the answers to the pre-treatment question at less than two months and at two months or more. The results showed a statistically significant increase in the pre-treatment score over time. The results indicated that the participants experienced a response shift, however, in the opposite direction from what was expected. This response shift suggests that the participants do not tend to exaggerate, but tend to forget how bad their level of fatigue was before taking Gene-Eden-VIR. The tendency to forget adds support to the statistical significance of the results in this study.
Latent infections are very common. The worldwide rates of HSV infection range from 65% to 90% [42] . The rate of EBV infection is about 95% of the world’s adult population. Epidemiological studies reported that the rate of HPV infection, over the lifetime of people in the USA between the ages of 15 and 50, is about 75% [43] . The rate of CMV infection in individuals in the USA at the age of 6 and older is 58.9%, and in certain countries it reaches 100% [44] . In most infected individuals, an efficient immune system controls the number of latent viruses [45] . However, when the environment changes, such as during stress, injury, aging, while taking certain medications, etc., the immune system weakens. As a result, Microcompetition with the higher number of latent viral particles overwhelms the cellular genetic system. One result of this intense Microcompetition is a feeling of fatigue.
5. Conclusion
This post marketing clinical study showed that treatment with Gene-Eden-VIR safely decreased the feeling of general, physical, and mental fatigue in individuals infected with a latent virus. Since most individuals are infected with a latent virus, health care practitioners should recommend Gene-Eden-VIR as a first line treatment for fatigue. The results of this post marketing clinical study support the Microcompetition with Foreign DNA theory.
NOTES
*Corresponding author.