Posiphen was shown in 72 healthy volunteers to be safe in SAD and 53 subjects in MAD and POM studies of 7–10 day administration up to levels determined as greater than fivefold the effective dose. This effective dose was determined both by comparing the extrapolated molar concentration in brain with the 50% effective concentration to inhibit APP in neuronal cultures, as well as from animal studies.13
The human pharmacokinetics of Posiphen/metabolites and extrapolation of data determined from rodents suggests that Posiphen readily enters the brain and achieves levels 6.8-fold higher than in plasma at steady-state, in accordance with its high lipophilicity (ClogP value 2.2). Its hydrophobicity and protein binding capacity (96% of Posiphen/ metabolites bind to brain proteins) likely limit levels of Posiphen/metabolites found in CSF of both rodents and MCI patients. Interestingly, Posiphen half life in CSF of MCI patients proved to be longer than its half life in plasma, >12 h versus approximately 5 h, respectively (). Consistent with its longer half life within the central nervous system, Posiphen's APP, τ and inflammation lowering activity lasted longer than the recorded 12 h of sampling. Recent studies in neuronal cultures indicate that Posiphen's APP lowering actions extend for numerous hours following wash-off and, additionally, are likewise maintained within the brain of transgenic AD mice for over 9 h after cessation of dosing (Sambamurti K, Medical University of S. Carolina, personal communication). The extended duration of Posiphen/metabolites in CSF/brain together with the prolonged inhibition of APP and τ expression may permit once a day dosing and is a focus of current studies.
Our early POM study in MCI patients focused on evaluating target engagement demonstrates that Posiphen lowers both sAPPα and sAPPβ in CSF, consistent with its preclinical actions13
and ability to inhibit the translation of APP mRNA via an iron response element within its 5′-untranslated region.14
The trend of a Posiphen-induced reduction in CSF Aβ42
in MCI subjects is likewise in line with its known action to inhibit APP synthesis, as Aβ42
is a downstream product and is in accordance with the described decline in CSF sAPPβ in MCI subjects as well as of Aβ42
levels in preclinical studies.13–15
A separate more limited analysis of Aβ40
(AlphaLisa) in CSF collected at 3 and 8 h prior to and following Posiphen administration in MCI subjects provided reduction trends of -32% and -37%, respectively. A caveat of early CNS target engagement investigational studies is the small patient number required to, on one hand, adequately demonstrate pharmacologically driven biological activity in the brain as a result of drug interaction with its intended target to provide proof-of-concept and, on the other hand, protect patients from exposure to potentially inactive or toxic drugs.29
In our Posiphen study, this was undertaken on five MCI subjects, one of whom withdrew, allowing biomarker analyses on four. However, as patients were used as their own controls at 0 day (prior to Posiphen treatment) and as test subjects following 10 days Posiphen treatment, the POM study design limited the potential effect of often large inter-subject variability30
thereby permitting statistical analyses on data derived from this small patient number. In this regard, individual patient data analyses are shown in . Clearly evident is the inter-subject difference in biomarker levels under naive (day 0) conditions (determined as the mean value ± SD of the nine timed samples across the 12 h sampling period). Evident also is the sometimes high variance around the mean biomarker value for each individual related to the time-dependent change (consistent with the circadian pattern reported by others31
) in biomarker levels over the 12 h study. Consistently across all individuals within , 10 day Posiphen administration lowered mean levels of sAPPα, sAPPβ, Aβ42
, t-τ, p-τ and C3, but not factor H. Importantly, the time-dependent analysis of biomarker levels within the same individual, by matching exact same times predrug versus postdrug (), allowed determination of Posiphen-induced differences in such a small patient number (N=4) in the presence of large inter-subject and time-dependent biomarker differences. Of significance, the pattern of the changes was remarkably alike between the different assays employed blindly to measure the same CSF analyte at different independent institutions (whether AlphaLisa vs MSD in the quantification of sAPPα and sAPPβ, or AlphaLisa vs Innogenetics for Aβ42
and t-τ). Albeit, the percent of the Posiphen-induced inhibition and variance differed between the assay techniques (); similar data deriving from the use of two independent assays provide a valuable level of cross-validation to help guard against unforeseen systematic errors. Clearly, without the potential to match predrug and postdrug time-dependent biomarker levels within the same patient, a far greater number of subjects would have been required to support statistical analyses. Nevertheless, a larger patient number, which is often limiting in early POM studies, would have provided greater statistical power to discriminate drug-induced biomarker actions, as would the inclusion of a placebo group.
Figure 5 Difference in biomarkers between day 0 (naive) and 10 day Posiphen (4×60 mg/day) administration. As the effect of Posiphen on the analysed biomarkers remained present over 12 h, we were able to calculate the mean of the nine samples (more ...)
Recent reports suggest that CSF elevations in sAPPα and, in particular, sAPPβ may be clinically useful and superior to assessing Aβ42
, in the early and differential diagnosis of incipient AD.32
Hence, as APP represents Posiphen's immediate target, CSF levels of sAPPα and sAPPβ, rather than, simply, Aβ42
, were measured and found to be elevated in our MCI patients compared with healthy controls (), in accordance with others.33
Posiphen's reduction in CSF sAPPα and sAPPβ in MCI patients brought their values in line with healthy controls. A preliminary analysis of Aβ42
, analysed by two techniques (), suggests reductions in the same order as sAPPα and sAPPβ.
Posiphen treatment led to statistically significant reductions in CSF levels of other key AD biomarkers, in particular t-τ and p-τ. As illustrated in and in accordance with others,35
CSF t-τ levels were elevated in our MCI patients versus healthy controls37
and were normalised by Posiphen. The relevance of these actions and mechanisms through which they are mediated are a focus of current studies. In this regard, resembling the action of Posiphen to impact the translational regulation of APP mRNA,14
τ can also be regulated at the level of its RNA stability,39
potentially by Posiphen. Alternatively, reductions in τ may be secondary to other actions or a combination of primary effects on translational regulation and secondary actions. Nevertheless, similar Posiphen-induced time-dependent reductions in τ have recently been found in neuronal cell cultures and preclinical AD models (Sambamurti K, personal communication). Posiphen, likewise, induced statistical declines in MCI CSF C3, a pro-inflammatory factor reportedly elevated in both AD transgenic mice41
and the CSF of AD subjects,42
together with key biomarkers (MCP-1, YKL-40) of microglial activation.24
In contrast, Posiphen treatment did not alter levels of the innate immune response protein sCD14 or factor H. Likewise, the decline in specific inflammatory markers may be a secondary effect to the described reductions of APP and τ.
In synopsis, our pharmacokinetic studies in humans and rodents permitted us to estimate levels of Posiphen/metabolites in human brain after Posiphen (4×60 mg/day, 10 days) to be in the order of 3.5 μM Posiphen, associated with the described biomarker changes. This drug level is greater than the determined 50% effective concentration of Posiphen to lower APP levels in neuronal cultures.13
Recent studies have demonstrated that each Posiphen metabolite, likewise, has APP lowering actions.43
We conclude that Posiphen appears to be a promising experimental drug for MCI and AD as it can effectively lower CSF levels of APP, its primary target in brain, and in addition lower t-τ, p-τ and key inflammatory markers, and may hence impact disease progression at a number of levels.