HUMAN TRIALS

Executive Summary

T.A. Sciences Pivotal 2005 Anti-Aging Trial of TA-65
April 18, 2007

Trial Summary

The Pivotal 2005 Anti-Aging Trial of TA-65 was a double blind, placebo controlled, 24 week study in which subjects consumed 2 or 4 tablets daily of a placebo control substance (placebo groups) for 12 weeks or 2 or 4 tablets daily of a TA-65 precursor molecule (TA-41) for 12 weeks (product groups). The product tablets each contained 10 mg of TA-41 (an Astragalus extract) along with other botanical extracts and excipients. The placebo control tablets were essentially indistinguishable from the product tablets in appearance and taste, even when the tablet was broken. The 12 week placebo or product use period was followed by a further 12 week follow-up period. To ascertain active substance in the blood (for compliance and to better understand the relationship between TA-41 and TA-65), analytical measurements TA-41 and TA-65 (the presumed major metabolite of TA-41) were conducted at 6 weeks and 12 weeks. Thirty six male subjects aged 60-85 who expressed an interest in reducing the signs and symptoms of aging and who reported a gradual decline in overall energy levels at the screening visit were recruited. Subjects were randomly assigned to the placebo group (n=6 taking two placebo tablets, one in the morning, one in the evening; n=6 taking 4 placebo tablets, two in the morning, two in the evening) or the product treatment group (n=12 taking two product tablets, one in morning, one in evening; n=12 taking 4 product tablets, 2 in morning, 2 in evening). Subjects were assessed at baseline and at 6 weeks, 12 weeks and 24 weeks from the first dose of product.

Statistical Methods

Statistical analyses of multiple economic, demographic and health and lifestyle-related measures show no indication that the subjects who were assigned to the product groups were significantly different at baseline than those who were assigned to the placebo groups.

The statistical tests performed on the data assign statistical significance to changes in study subjects that occur in those that were on the product compared to those on placebo. Because the separate groups were relatively small and initial analyses did not show a clear dose-related pattern in the two treatment groups (20mg TA-41 vs 40mg TA-41), the two placebo groups were combined into a single placebo group, and the two product groups were combined into a single product group for all comparisons, thus increasing the power of the study. Because of notable variation in baseline values of study subjects for most of the variables tested, we specifically assessed the changes from baseline (up or down) for each subject at each of the 6, 12, and 24 week timepoints. For some measures pre-treatment values had been measured twice, and the mean of those values is used as a baseline for comparison. The means of the individual changes at each timepoint in the treatment versus the placebo groups are compared using a Student's t-test statistic. The alternative method of taking the changes from the baseline mean for the group means at each timepoint is less sensitive in this small sample. We want to be able to detect individual improvements against baseline values allowing for large differences between individuals. The t-test yields a p-value, namely the probability that the difference in the set of changes from baseline for each subject in the treatment group versus the set of changes for each subject in the control group is due to chance. A two-tailed t-test was utilized. Small p-values indicate that differences are significant, i.e. likely not due to chance.

We systematically took multiple approaches to the data set since this was the first human study with a telomerase activator and there was very little prior knowledge relevant to when peak responses might occur, and whether we should expect significant variations in the kinetic profile of responses (during or after treatment) between individuals, or between different tissue or organ systems. A complete list of the analyses conducted is described in the full study report (Appendix A). For this executive summary, the t-test statistical method, consistent with the original intent of the study design, was used for selection of the key variables showing apparent positive structure/function changes in the product group versus the placebo group. This method addressed the following question:

Is there consistent difference in how individuals changed from baseline between the product and placebo groups during the study period (i.e. at 6 weeks, 12 and 24 weeks)?

For each variable, analysis of each timepoint was done separately. Given the relative small size of the study, it was not expected that data at each timepoint would necessarily reach statistical significance, but if a positive trend emerged and at least one timepoint reached statistical significance, changes in this variable were deemed significant.

For the key functional groups we found that significant p-values occurred in many or all of the study variables related to the finding. No such co-occurrence of p-values is observed in other parts of the study. The frequency of significant p-values far exceeds what would be seen by chance.

Because of limited study size, and consistent with general practice in small dietary supplement studies, it was determined a priori that all t-tests that have a p-value below 0.2 would be considered: A p-value greater than 0.1 but less than or equal to 0.2, is identified as a trend, while p-values less than or equal to 0.1 are defined significant. Although we are primarily looking for indications of changes to be validated in larger scale studies, we are calling special attention to those variables or summary variables that reach p-values less than 0.05, the generally accepted level of significance for larger scientific studies.

Summary of significant changes

Where we see trends or significant changes in study variables, they cluster together in functionally meaningful groups. The major groups of benefits were seen as:

1. Apparent improvement in certain immune system measures.
2. Apparent improvement in eye sight.
3. Apparent improvement in certain sexual function measures.
4. Apparent improvement in certain skin properties.

We believe that these changes are meaningful since a positive picture emerges both statistically and in terms of the magnitude of change with more than one variable in each of these areas. While these significant positive changes were seen in certain study variables for the treatment group compared to the control group, there were no clinically significant adverse effects attributed to the product (see Summary of Safety Assessment below). In some cases, the control group showed a decline from baseline at one or more timepoints while the treatment group showed improvements from baseline. The placebo tablets contained wheat germ and inert excipients, thus it is unlikely that the decline from baseline seen for some variables represents a negative property of the placebo tablet. More likely a decline from baseline represents an effect due to some uncontrolled variable (e.g. season), or it could be due to random variations which periodically would show negative trends in either the placebo or product groups.

A. Immune System

There were three types of analyses addressing the immune system:

• lymphocyte proliferation in response to antigenic stimulation (an increase in this variable was considered a positive change)
• inflammatory cytokine levels (a decrease in this variable was considered a positive change)
• numeric analysis of certain white blood cell subsets associated with cellular aging (an increase in a normal cell subset known to decline with age was considered a positive change, while a decline in the number of cells showing markers of aging was considered a positive change)

There were clear positive trends for variables in two of the three areas investigated: lymphocyte proliferation, and natural killer cell counts (Fig. 1). There were no consistent, significant trends in other variables tested in the immune panel.

Lymphocyte Proliferation

Lymphocyte proliferation was measured at baseline and at 6, 12, and 24 weeks using a standard assay in which white blood cells are exposed to a potent plant antigen called phytohemaglutinin (PHA) which primarily activates T lymphocytes. The amount of new DNA synthesis following PHA stimulation is measured, reflecting the proliferative (cell division) response.

The consistently greater increase in lymphocyte proliferation under conditions of strong stimulation with PHA suggests that TA-65 improves the proliferative response of the immune system to foreign agents. T lymphocytes are responsible for the cellular "adaptive" immune response in the body against foreign invaders. The strong proliferation response of antigen-specific T lymphocytes is critical to mounting a strong immune response against foreign material which the body has been exposed to in the past. It is known that various immune functions decline as we age and much of this decline is associated with T lymphocyte aging (1-7). In particular, there is a significant body of literature on the role of telomere shortening in lymphocyte aging and its possible link to reduced cell division capacity and reduced ability to respond functionally to antigenic stimulation (1, 7-13). This loss of cell division capacity and functional response of immune cells can be corrected, in part, by activating telomerase in cultured human cells using the telomerase gene (8, 11, 13). The data presented in Figure 1 provide the first demonstration that a small molecule natural product which activates telomerase in human cells in culture may have the ability to improve the immunological response of T cells in elderly men.

The magnitude of the TA-65 response in the PHA assay appears significant. The mean increase in proliferative response for the product group at 24 weeks was about 53% over the baseline response. The general increase in both the product and placebo groups over time might relate to seasonal changes, but at 24 weeks the difference in the individual responses in the product group was highly statistically significant from that in the placebo group (p=0.0049). At the earlier time points (6 and 12 weeks), the product group showed greater increases over baseline compared to the placebo group, but these differences did not reach statistical significance, perhaps due to the small sample size. The greater magnitude of effect of TA-65 at 24 weeks (12 weeks after discontinuation of product) compared to the effect at 6 or 12 weeks was not expected. It may be that the potential molecular effects of TA-65, for example on telomere length (which was not measured in this study), might take weeks or months to manifest itself at the cell and tissue level.

Natural Killer (NK) Cells

NK cells are specialized lymphocytes belonging to the "innate immunity" arm of our immune system. These cells can immediately mount a killing response against certain tumor cells, foreign cells from infections, or cells infected with a foreign agent such as a virus, without having been previously exposed. It is known that NK cell function declines with age and is associated with increased morbidity and a loss in life satisfaction (14-16). Recently it has been shown that, as with other hematopoietic cell lineages, telomere length in NK cells declines with age in humans, especially in very old individuals (17, 18), suggesting that telomere-dependent aging of NK cells may account for the partial loss of NK function in the elderly. In this study, NK cells were measured by the number of CD3-/CD57+ cells at baseline and at 12 weeks. There was no assessment at the 6 or 24 week timepoints.

The treatment group had an average increase in NK cell numbers of about 14% at 12 weeks, while the NK cell number in the placebo group dropped about 10% in the same time period (Fig. 1). As indicated in the introductory section, why the placebo group sometimes shows a decline from baseline is not known, but may represent seasonal effects and/or random fluctuations. The difference in treatment versus control groups had a p-value of 0.077, which for this study was considered significant. The improvement in NK cell numbers in the elderly men exposed to TA-65 versus the control group of men suggests they could have an improved immune response against foreign or abnormal cells in the body.

Overall, these data on improved proliferative response of T cells and an increased number of NK cells in the treatment group suggest that TA-65 has a structure/function effect which could translate into an improved immune system in elderly men. Since women show similar signs of immune system aging with time, it is likely that their immune system could also be strengthened by TA-65.

B. Vision

It is well established that vision declines with age for a variety of reasons related to deterioration in structure and function of multiple compartments within the eye, including the lens, retina, and, importantly, the blood vessels (vasculature) supporting the eye tissues. Only recently has it been appreciated that telomere-related cellular aging in tissues of the eye and elsewhere in the body may play a causal role in declining eyesight (19-23). The potential benefits of telomerase activation on aging vasculature (see section on Sexual Function) could be important to improvements throughout the body, since optimal blood flow blood and vessel density contribute to proper structure and function of all tissues.

In this study, a standard visual acuity test (assessment of distance at which a person with normal vision would have comparable acuity to the tested individual viewing symbols at 20 feet) was conducted at baseline and at 6, 12, and 24 weeks. Tests with only the left or right eye open, and with both eyes open, were conducted. Improvements in the product group compared to control group were seen with the left eye and with both eyes, and these data were combined in the vision improvement summary (Figure 2). The average improvement over baseline in the product group was greater than that in the control groups at all 3 timepoints tested. The difference at 6 weeks was statistically significant (p=0.075) and at 12 weeks the data suggested a trend in improvement (p=0.12). Although the product group performed better than the control group at 24 weeks, this difference was not significant (p=0.47). The apparent decline in effect of TA-65 at 24 weeks compared to 6 or 12 weeks could be due to natural fluctuations in measurements (random variations) or it may be due to the treatment group being off TA-65 since week 12.

Overall the data suggest that TA-65 has an effect on structure/function which causes improved vision in elderly men. This small study does not definitively prove that TA-65 improves vision in the elderly, but if real, the mechanism could involve improvements in metabolism or vasculature in the eye, or improvements in function of lens epithelial or retinal cells. Since similar deterioration of vision occurs with age in women, it is possible that vision in women could also be improved by TA-65.

C. Skin

As with vision and the immune system, there has been significant research on the role of cellular aging and telomere attrition in several types of skin cells, including keratinocytes, fibroblasts, microvascular endothelial cells, and melanocytes (21-31). Moreover, for all of these cell types, studies have shown that activation of telomerase by genetic means has improved replicative capacity and/or function of aging or stressed skin cells (reviewed in references 32, 33).

In this study, skin quality was assessed at baseline and at 6, 12, and 24 weeks by the subjects themselves using a questionnaire. In 8 of 10 categories queried, there were consistent improvements reported in the product group over baseline compared to changes reported in the control group. These categories were combined into a single summary variable and the data shown in Figure 3. Although these reports were not based on objective measurements of skin structure and function, the individuals were not aware of whether they were taking the product or the placebo tablets, and hence the consistent improvement in skin assessments in the product group across multiple measures suggests a significant effect of TA-65 on skin. As was seen in the vision scores, the average improvement over baseline in the product group was greater than that in the control groups at all 3 timepoints tested. The difference at 6 weeks was highly significant (p=0.037), while the data at 12 and 24 weeks suggested a trend at best (p=0.22 and 0.17, respectively).

Overall these results suggest that TA-65 improves the structure and/or function of skin in elderly men. Since similar age-related deterioration of skin occurs with age in women, it is possible that female skin could also be improved by TA-65.

D. Sexual Function

The decline in sexual function with age in men is complex and likely involves neurological and hormonal systems as well as proximal changes in penile tissue, particularly vascular tone regulation in the corpus cavernosum which is required for an erection. Many studies have linked telomere biology to age-related decline in the central nervous system (35-38), hormonal balance (39-42), and vascular function (28, 29, 36). Most importantly it has been demonstrated that aging of endothelial cells (the inner lining of blood vessels) is associated with telomere loss and that genetic activation of telomerase can dramatically increase the replicative capacity and improve the function of aging endothelial cells (28, 29, 43). In particular, the nitric oxide signaling pathway of endothelial cells, which is important for vascular tone in the corpus cavernosum and erectile function (44), is compromised with age, and published studies show that telomerase activation with gene transduction helps restore nitric oxide synthase expression and nitric oxide signaling (45-46).

In this study, sexual function was self-assessed at baseline and at 6, 12, and 24 weeks by the subjects using a standard questionnaire. In two categories ("ability to have intercourse", and "satisfaction") there were consistent improvements reported in the product group over baseline compared to changes reported in the control group. These categories were combined into a single summary variable and the data shown in Figure 4. The average improvement over baseline in the product group was greater than that in the control groups at all 3 timepoints tested. The differences at 12 and 24 weeks were significant (p=0.072 and 0.033, respectively), while the data at 6 weeks showed a trend (p=0.13).

Overall these results suggest that TA-65 improves sexual function in elderly men. Whether TA-65 would improve sexual function in females should be evaluated in a separate study.

To read a Summary of the Safety Assessment of the Trial, click here .

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