In-vitro results
Using the LPO-specific probe BODIPY C11, PSP MSCs had a significantly higher rate of LPO compared to controls. After a 72-hour incubation of the cell lines with RT001, PSP MSCs returned to normal levels, while PSP MSCs incubated with non-deuterated linoleic acid ester (H2-LA) remained elevated (Figure 1, Panel A). The time course of lipid peroxidation for the various MSCs and treatments is displayed in Figure 1, Panel B.
Glutathione levels were measured using MCB. The MCB intensity was reduced in PSP MSCs compared to HC. After incubation with RT001, glutathione levels were restored to HC levels, while glutathione levels remained low in PSP MSCs after incubation with H2-LA (Figure 1, Panel C. Representative images of these cells are shown in Figure 1, Panel D.
Panel A) Bar chart quantification of the efficacy of RT001 on the rate of lipid peroxidation using C11-Bodipy (PSP alone vs. PSP + RT001, p < 0.0001). Panel B) Representative time course traces of lipid peroxidation in MSCs derived from HC (light green), and PSP (red), PSP treated with RT001 (orange), and PSP treated with H2-LA (dark green), respectively. Panel C) Measurements of monochlorobimane (MCB) fluorescence intensity as an indicator of glutathione (GSH) levels (PSP alone vs. PSP + RT001, p < 0.0001). Panel D) Representative images showing MCB (GSH) fluorescence intensity for HC, PSP, and PSP + RT001. MCB intensity is reduced in PSP compared to HC, but are restored after incubation with RT001. The coarse dash lines approximate the cell borders of an individual MSC (fine dash line). Data are represented as mean ± SEM. Total number of cells per well n = 10 – 50 from 3 – 6 culturing wells. All experiments were repeated 2 – 3 times (N, independent culturing conditions). *p<0.05, **p<0.001, ***p<0.0001.
The fluorescence intensity of TMRM was increased in the PSP MSCs relative to HC MSCs, indicating in increase in the Δψm. This increase remained elevated when the cells were incubated with H2-LA, but Δψm normalized after RT001 (Figure 2, Panels A and B). The changes seen in the Δψm were also seen in the fluorescence of MitoTrackerCM-H2Xros. Fluorescence intensity of MitoTrackerCM-H2Xros for the PSP MSCs was increased more than 2.5 times HC at baseline, indicating an increase in mitochondrial ROS. Incubation with H2-LA reduced mitochondrial ROS generation slightly, but RT001 reduced mitochondrial ROS back to near normal levels (Figure 2, Panels C and D). Fluorescence intensity with Pico Green exhibited an inverse correlation with the other studies. PicoGreen fluorescence was decreased at baseline for the PSP MSCs relative to HC, indicating a reduced amount of mitochondrial DNA. After incubation with H2-LA, fluorescence increased slightly, but was far more pronounced for the RT001 incubated cells. In addition to increased mitochondrial DNA amount, the structure and number of mitochondria were also increased by RT001 treatment (Figure 2, Panels E and F).
Panel A) Histogram demonstrating the mitochondrial membrane potential (Δψm) measured using the fluorescence intensity of TMRM (tetramethylrhodamine). Δψm was increased in PSP MSCs compared to HC. Δψm was reduced after incubation with RT001, but not after incubation with H2-LA (p=0.0009). Panel B) Representative images depicting the mitochondrial shape, distribution, and fluorescence intensity at baseline for HC and PSP MSCs (fine dash line represents the approximate cell border of a MSC). Panel C) Quantitative histogram of MitoTrackerCM-H2Xros fluorescence intensity shows baseline elevations in ROS in the PSP MSCs are reduced to near normal levels after incubation with RT001 (p<0.0001), but not after incubation with H2-LA (p=0.0801). Panel D) MitoTrackerCM-H2Xros fluorescence over time for HC, baseline PSP, PSP + RT001, and PSP +H2-LA. Baseline fluorescence elevations for the PSP MSCs over HC MSCs were restored to near normal after RT001, but not after H2-LA incubation. e Representative images of the mitochondrial DNA content of PSP MSCs at baseline (top panel) and after incubation with RT001 (bottom panel): note the difference in the extranuclear distribution of the PicoGreen fluorescence. A fine dash line represents the approximate cell border of a MSC. Quantification bar chart of the PicoGreen Intensity as a measure of mitochondrial DNA content. Baseline (orange columns) reductions in mitochondrial DNA were seen in the PSP MSCs (middle and right histograms). Incubation with H2-LA (middle histogram) resulted in a small increase in mitochondrial DNA (middle histogram, green column; p=0.0689), while RT001 restored PSP MSCs to normal levels (right histogram, green column; p=0.0010). Data are represented as mean ± SEM. Total number of cells per well n = 10 – 50 from 3 – 6 culturing wells. All experiments were repeated 2 – 3 times (N, independent culturing conditions). *p<0.05, **p<0.001, ***p<0.0001.
Clinical results
Baseline demographic information for the 3 subjects is displayed in Table 1.
Table 1. Baseline characteristics for the 3 subjects at the onset of treatment
Characteristic
|
Subject Number
|
1
|
2
|
3
|
Age (years)
|
66
|
73
|
74
|
Sex
|
Male
|
Male
|
Female
|
Pre-treatment symptom duration (years)
|
6
|
3
|
2
|
PSP diagnosis
|
Probable
|
Possible
|
Probable
|
Baseline PSPRS
|
17
|
12
|
13
|
Baseline UPDRS
|
44
|
36
|
21
|
The linear regression slopes of the PSPRS and UPDRS scores for the 3 subjects were plotted against those obtained from disease progression predicted by previous longitudinal studies of untreated PSP patients. Figure 3 shows the slope of the PSPRS changed from the historical decline of 0.91 points/month to a mean of decline of 0.16 points/month (+/- 0.23 SEM). The UPDRS slope changed from an expected increase of 0.95 points/month to an average increase in score of 0.28 points/month (+/- 0.41 SEM).
Pharmacokinetics
Mean plasma and RBC membrane levels of drug were 21% and 19% of total linoleic acid. Levels of di-deuterated arachidonic acid in both plasma and RBC also increased, indicating normal enzymatic processing of the stabilized LA into stabilized AA.
Safety
Overall, RT001 was well tolerated. One serious adverse event occurred (cerebrovascular event) that was not drug-related.