In vitro examination of the process-specific mechanism of CLR01
As stated above, the mechanism by which CLR01 remodels the assembly of amyloidogenic proteins into non-toxic assemblies that can be degraded by normal clearance mechanisms is by its specific binding to Lys residues. The mechanism is “process-specific” because it is postulated to affect only the aberrant assembly of proteins that leads to toxic oligomers and aggregates, but not normal protein structure, function, or assembly as happens, e.g., in tubulin polymerization. To test whether this indeed is the case, we examined the effect of CLR01 on tubulin polymerization [21, 22]. Three mg/ml (~18 μM) porcine brain tubulin, which contains 3.8% Lys and 4.8% Arg, was allowed to polymerize in the absence or presence of CLR01 concentrations ranging from 10–1,000 μM.
In the absence of CLR01 or in the presence of up to 300 μM of the compound, the change in turbidity followed a typical sigmoidal curve, starting at 0.05-0.09 absorbance units (Figure 2). The absorbance remained unchanged for the first 10–15 minutes, which is a typical lag phase in this reaction, and then increased gradually up to ~60 min, at which point the rate of increase began to decline, and the reaction was followed for another 10 min. The only concentration at which significant modulation of the polymerization was observed was 1,000 μM (Figure 2, blue curve), i.e., at a tubulin:CLR01 concentration ratio ~1:55. At this high ratio, a high absorbance, 0.15, was observed immediately, followed by a slight gradual decline during the lag phase. Then, the absorbance began to increase for 30 min, followed by a slow decline for the rest of the experiment. One interpretation of these data is that at the high concentration used, 1,000 μM, binding of CLR01 to tubulin induced immediate self-assembly into irregular aggregates. Similar immediate induction of self-assembly was observed with 4 of the 9 amyloidogenic proteins tested by Sinha et al. [3], suggesting that this reaction occurs with some, but not all proteins. In all the cases studied by Sinha et al., these aggregates were non-amyloidogenic and non-toxic.
Presumably, following the immediate aggregation in the presence of 1,000 μM CLR01, the irregular tubulin aggregates observed at t = 0 partially disassembled as the polymerization reaction progressed, between 10–30 min. At that point, the high CLR01 concentration appeared to interfere with the polymerization reaction and the tubulin polymers gradually disassembled. Validation of this interpretation will require further investigation, yet it was not the focus of the current study.
The motivation for this experiment was to test whether the concentration of CLR01 needed to interfere with a controlled self-assembly process was substantially different from that required for modulation of aberrant self-assembly, which was found indeed to be the case. Most of the protein:CLR01 concentration ratios needed for inhibition of amyloidogenic protein aggregation were in the range 1:1–1:3 [3], compared to the 1:55 tubulin:CLR01 concentration ratio at which disruption of tubulin polymerization was observed. These results support the specificity of CLR01 for inhibition of aberrant aggregation as opposed to controlled polymerization.
CLR01 safety
If CLR01 indeed operates by a process-specific mechanism and remodels the abnormal aggregation of amyloidogenic proteins at substantially lower concentrations than concentrations that would perturb normal physiological processes, one would expect the compound to have a high therapeutic index. To calculate the therapeutic index, a lethal dose must be reached. The in vitro data described above suggested that disruption of tubulin polymerization occurs at concentration ratios 20–50 times higher than those needed for inhibition of aggregation of amyloidogenic proteins. In addition, cell culture experiments indicated that CLR01 began to show toxicity at concentrations 1–3 orders of magnitude higher than those required for inhibition of toxicity by different amyloidogenic proteins [3, 4]. The next rational step was to test the safety margin of CLR01 in vivo. Based on the in vitro and cell culture data, we expected that 100 mg/kg would be lethal to mice and therefore used it as the highest dose in our safety-evaluation experiments. We assessed the safety of CLR01 in 2-m old, male, WT mice either 24 h following a single IP injection of 10 or 100 mg/kg (acute administration) or after daily IP injection of 3 or 10 mg/kg for 30 days (chronic administration). Following euthanasia, serum was collected for chemical analysis and tissues were harvested for histopathology evaluation.
All CLR01-treated groups, except for the 100-mg/kg acute-administration group, behaved indistinguishably from control mice in terms of levels and type of activity and grooming. The administration of 100-mg/kg CLR01 caused obvious signs of distress immediately, which lasted for ~30 min following the injection. For most mice, activity level decreased and eyelids became droopy. Some of the mice exhibited arching of the back, sporadic gasping, lying down, dragging one leg, and twitching. These signs of distress diminished after the first 30 min, at which point the mice resumed grooming and sitting on hind legs. Some mice showed decreased activity and droopy eyelids for up to 2 h following the injection. No symptoms of severe toxicity, as defined by the UCLA DLAM veterinarians, were observed for any mice, including bruising, bleeding, pale mucous membranes or extremities, diarrhea, paralysis, tachypnea or dyspnea, or abdominal distension.
Liver, kidney, spleen, heart, lung, and brain were collected for histopathology analysis. Tissue samples from heart, lung, spleen, and brain of all acutely CLR01-administered mice were indistinguishable from those of control mice. In all 100-mg/kg-dosed mice and one of eight 10-mg/kg-dosed mice of the acute-administration groups, liver degeneration and necrosis was detected in centrilobular and midlobular regions (Figure 3). Zonal nature of liver toxicity is common in drug-toxicity studies and was expected in the high-dose group.
The fact that all the mice in the high-dose group survived meant that the actual therapeutic index could not be calculated because contrary to our expectation, 100 mg/kg was under the lethal dose. However, we considered the observation of obvious liver toxicity at this high dose as sufficient for determining the maximal dose in future efficacy experiments and therefore did not treat mice with higher doses. Rather, we conducted next a 30-day, chronic-toxicity experiment in which mice were administered IP either 3 or 10 mg/kg/day of CLR01. Because one mouse of the eight used in the 10-mg/kg acute-administration group showed signs of liver toxicity, 10 mg/kg/day was chosen to be the highest dose in this experiment.
Heart, lung, spleen, and brain from both chronically CLR01-treated groups of mice were indistinguishable from vehicle-treated mice and were free of signs of malformation, degeneration, necrosis, or inflammation within normal variability among mice. A few mice in the 3-mg/kg group showed signs of mild-to-moderate multifocal extramedullary hematopoiesis in the liver. The consulting veterinary pathologist concluded that this was possibly immune-stimulated but not pathogenic. Mild pancreatitis also was observed in one of the mice showing liver hematopoiesis and one additional mouse in the 3-mg/kg group. In contrast, no signs of tissue pathology or liver necrosis were detected in any of the mice in the 10-mg/kg dosed group. Thus, it is unlikely that the hematopoiesis or inflammation found in the low-dose group were related to the CLR01 treatment.
Serum chemical analysis mainly consisted of tests of renal and liver function (Table 1). No significant differences were observed between the control and low-dose groups in either the acute-administration or chronic-administration experiments. The acute-administration, 100-mg/kg group showed significant increase in alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, and a significant decrease in cholesterol compared to both the control group and what is considered a normal range (UCLA DLAM, modified [23]). All of these changes are consistent with acute liver injury. Glucose levels were significantly lower in the 100-mg/kg acute-administration group than in the control group, but were within the normal range. Production of glucose is often the last function to be lost in liver damage. Other changes indicating liver damage, including changes in concentrations of albumin, alkaline phosphatase, or total bilirubin, were not observed. In the chronic-administration experiment, the only significant serum-chemistry difference observed was ~40% reduction in blood cholesterol in the 10-mg/kg group compared to the control group. The cholesterol level was within the normal range.
Pharmacokinetics of CLR01 in vivo
The plasma concentration of CLR01 was measured by LC-MS in 2-m old WT mice following administration by a SC or IV injection or by oral gavage. The SC bioavailability was found to be identical, within experimental error, to the IV administration, which was considered as 100% bioavailable (Figure 4). In both routes, ~30% of the administered dose was detected in the blood at the earliest time point measured – 20 min, and the plasma half-life was found to be ~2.5 h. Approximately 5% of the initial CLR01 levels were found in the plasma 8 h following either SC or IV administration. Oral bioavailability was negligible, suggesting that CLR01 gets metabolized in the gastrointestinal tract and/or does not pass from the gut to the blood.
Next, we asked what percentage of the administered CLR01 penetrates through the BBB and gets into the CNS. Our first attempt was to measure CLR01 in brain extracts using LC-MS. However, this proved to be difficult. Due to the multiple negative charges of CLR01, its partial protonation at physiologic pH, and the presence of various counter-ions in biological fluids, the MS signal splits into multiple peaks resulting in low signal-to-noise ratio. The difficulty to observe the CLR01 signal in brain extracts using LC-MS suggested that the concentration was low and detection would necessitate considerable optimization of the extraction and LC-MS methods, which would require substantial effort and high costs. Therefore, we decided to test first whether CLR01 could be found in the CNS by using a radiolabeled derivative of the compound.
As the permeability of the BBB has been shown to be dependent on age and morbidity, and in particular to be increased in AD [17] and in mouse models of AD [24, 25], we assessed how age and disease progression affected the brain penetration of CLR01 by using WT and 3×Tg mice at three different ages. The 3×Tg model was chosen because it was used in a previous study, in which CLR01 was found to reduce AD-like pathology in the brain [5]. Mouse ages were chosen to correspond with: 1) a stage before Aβ burden and cognitive deficits are found at 2-m of age [14, 26]; 2) a stage with mild-to-moderate plaque and tangle pathology but with observable memory deficits at 12-m of age [14, 27]; and 3) a stage of abundant plaque and tangle pathology with consistent behavioral deficits at 22-m of age [28]. Mice were administered 3H-CLR01 IV, blood and brain were collected at time points between 0.5 - 72 h following CLR01 administration, and radioactivity levels were measured by liquid scintillation counting. Radioactivity is presented as CPM/g of brain or CPM/ml of blood.
To correct for the radioactivity associated with blood-borne 3H-CLR01 in the brain vasculature, we performed both perfusion and subtraction analyses. In perfusion experiments, WT and 3×Tg mice at each of the three ages analyzed (n = 3 per group) were perfused with phosphate buffered saline following euthanasia. Perfusion lasted for either 5 min or until the liver changed color from a red to yellow, whichever was longer. In other experiments, mice were not perfused, but radioactivity associated with 10 μl of blood per g of brain [29, 30] was calculated based on brain weight and blood radioactivity levels and subtracted from brain radioactivity levels. At 1 h post injection, perfusion-corrected brain values were statistically similar to subtraction-corrected brain values (Figure 5). Due to difficulties associated with the perfusion analysis, specifically liver color being used as an indirect readout of brain perfusion level, and because including a perfusion step could increase variability among experiments, the rest of the experiments utilized the subtraction method, which is a common practice in BBB-permeability studies [29, 30].
At 0.5 h following injection, blood radioactivity levels in 12-m old mice were 39 ± 13% and 40 ± 6% of the injected levels, for WT and 3×Tg mice, respectively. These values were in agreement with the CLR01 concentration levels detected in plasma by LC-MS. About 5 - 10% of the radioactivity observed at time 0.5 h remained in the blood after 8 h (Figure 6).
Brain-radioactivity levels, calculated as a percentage of blood-radioactivity levels (CPM/g)/(CPM/ml) at 1 h following the injection ranged from 0.86–3.09% depending on age and genotype (WT versus 3×Tg, Figure 7). Analysis of brain penetration levels at 1 h by absence or presence of AD transgenes and by age showed a statistically significant effect of age but not of genotype. Interestingly however, 2-m old 3×Tg mice significantly differed from 12-m and 24-m old 3×Tg mice (2-m: 3.09 ± 0.55%; 12-m: 1.43 ± 0.17%; 24-m: 1.45 ± 0.28%; p < 0.05), whereas in the WT group, the only significant difference was between the 2-m and 24-m old mice (2-m: 2.68 ± 0.31%; 12-m: 2.11 ± 0.69%; 24-m: 0.86 ± 0.17%; p < 0.05). This suggests that changes in BBB permeability occur earlier and more sharply in 3×Tg mice compared to WT mice.
Surprisingly, although blood radioactivity levels declined rapidly (Figure 6), the radioactivity levels measured in the brain did not change significantly up to 72 h post-injection (Figure 8). Brain radioactivity levels were insensitive to genotype or time after injection and thus the 24-h time point was assessed only in the 22-m old mice (both 3×Tg and WT) and the 72-h time point was assessed only in the 22-m old WT mice. Differences were statistically insignificant and within experimental error.
To explore further the mechanics of CLR01 transport across the BBB, we asked whether the transport system was saturated. To answer this question, we injected 5-times the amount of total CLR01, keeping the ratio of 3H-CLR01:CLR01 at 1:9, into 22-m old WT mice. This experiment resulted on average, in 5-times the absolute amount of radioactivity detected in the brain. The percentage of brain penetration at 1 h following the injection did not change significantly (1× CLR01 brain penetration: 0.86 ± 0.30% of blood; 5× CLR01 brain penetration: 0.97 ± 0.28% of blood; Figure 8). This result suggests that the transport mechanism, whether active or passive, is concentration-dependent because there was an increase in the absolute value but not the relative value of CLR01 entering the brain.
To begin to explore whether additional dosing would increase the effective CLR01 concentration in the brain, we injected 22-m old WT mice twice over two equal time intervals and compared brain levels to mice that received one injection. On average, over the 1-, 3-, and 8-h time points measured, the amount of radioactivity found in the brain following the double-injection was twice the amount measured following the single-injection protocol (1 h: 3.3× compared to one injection, 3 h: 1.6×, 8 h: 1.9×; Figure 8). These data suggest that upon continuous dosing, as with the SC osmotic mini-pumps used in our previous efficacy study [5], CLR01 could reach sufficiently high brain concentration levels to inhibit Aβ aggregation even though the dose was relatively low — 40 μg/kg/day – when brain penetration levels are taken into account (see Discussion).
In vitro catabolism of CLR01
The BBB permeability experiments described above used radioactivity as an indirect readout of CLR01 concentration levels, which could have reflected the parent compound, CLR01 itself, or its metabolites. The question of the source of radioactivity seemed particularly important in view of the surprising persistence of radioactivity attributed to CLR01 in the brain. The most likely metabolism of CLR01 is cleavage of one or both phosphate groups resulting in monophosphate and hydroquinone derivatives, respectively (Figure 9). Each such dephosphorylation would decrease the polarity of the compound and increase its potential partition into the lipophilic brain parenchyma environment relative to the blood. In particular, the hydroquinone product is insoluble in aqueous solutions, in contrast to CLR01 and its monophosphate metabolite, which are soluble at millimolar concentrations. Thus, double dephosphorylation could result in precipitation and accumulation of the hydroquinone in the brain, potentially leading to misinterpretation of the BBB permeability data. Complete analysis of CLR01 metabolism in the brain was beyond the scope of the study described here. However, to evaluate the potential for dephosphorylation, we incubated CLR01 in vitro with ALP or brain extracts and measured the release of inorganic phosphate.
ALP is a widely distributed plasma enzyme found in many tissues which can be released into body fluids [31]. The enzyme received its name because it shows optimal activity at pH ~9. There are four isoforms of ALP: intestinal, placental, germ cell, and tissue non-specific. All four isoforms are non-specific enzymes that catalyze the hydrolysis of a wide range of phosphate esters [32]. Tissue non-specific ALP concentration levels increase in both brain and plasma of patients with familial or sporadic AD relative to age-matched healthy individuals [33], possibly as a compensatory mechanism because the enzyme catalyzes tau dephosphorylation [34].
Because of its promiscuous hydrolysis activity, we tested whether calf intestinal ALP catalyzed CLR01 dephosphorylation by incubating the molecular tweezer with ALP and comparing the amount of inorganic phosphate released to a standard curve obtained by incubating ALP with increasing concentrations of a common substrate, p-nitrophenylphosphate. This standard curve had a detection sensitivity limit of 5 nmol. Incubation of up to 100 nmol CLR01 with ALP resulted in undetectable levels of inorganic phosphate, suggesting that despite its promiscuity, ALP did not catalyze dephosphorylation of CLR01.
To test whether CLR01 dephosphorylation might be catalyzed by brain phosphatases other than ALP, we incubated 50 nmol CLR01 with 1.5 mg of mouse-brain homogenate. The brain homogenate dephosphorylated the positive control substrate, p-nitrophenylphosphate, at 99 - 130% of the activity of 0.8 enzymatic units of ALP. In contrast, similarly to the reaction with ALP, no release of inorganic phosphate was detected when the brain homogenates were incubated with CLR01 under the same conditions. Based on these results, dephosphorylation of CLR01 likely did not happen in our BBB permeability experiments and the radioactivity measured in mouse brains plausibly reflected CLR01 itself.