I have been anxiously awaiting this moment, the time in which I can write a blog post on our pilot study with Vium, formerly Mousera. Perlstein Lab had an opportunity to work with Vium starting about one year prior to their out-of-stealth launch, and I am itching to tell you about their company and the pilot project we conducted.
As we were approaching the end of our NPC primary screens, we started the process of looking for CROs to conduct mouse studies (PK, tox and efficacy) for the NPC disease R&D pipeline. During that process, Ethan was connected to Vium’s cofounder Tim Robertson by Cindy Wu, cofounder of Experiment. At our first meeting with their team, it was immediately apparent that we are like-minded companies with similar goals. While our technologies are very different, Vium complements our goals in making the drug discovery processes more efficient. Through digitizing mouse experiments, they are lowering the cost of mouse studies, reducing the animal handling during breeding and experimentation, and incorporating sensor and computer vision technologies to capture metrics that throughout the history of mouse experimentation were previously unavailable. I encourage you to check out the details of their technology by contacting them directly, but bottom line, this allows for mice to be studied in a more natural environment with minimal human handling affecting results and importantly for our needs, higher resolution on characterization of disease models, including the tracking and potential discovery of early and novel endpoints.
Being excited at the opportunity to work with Vium while they were in beta, we decided it would be best to run a pilot study through their novel platform to replicate results in the literature and potentially uncover novel metrics to monitor disease. Since the first disease we are working on in our high-throughput drug discovery pipeline is Niemann-Pick Type C, we wanted to validate that mouse model with Vium’s technologies. We designed a pilot study with the assistance of Vium’s team to test 2-hydroxypropyl-beta-cyclodextrin, currently in clinical trial, in the NPC NIH mouse model, BALB/c Npc1m1n (Npc1nih/nih). Breeding pairs were obtained from The Jackson Laboratory and sent to Vium for colony generation.
So what did our study look like? Well we quickly found out that breeding the NPC NIH mice did not yield expected mendelian ratios, however this has since been resolved, meaning our homozygous animal population at that time was small. Although, with each round of breeding, the number of homozygous animals per litter increased, and we obtained enough homozygous NPC animals for our study. Our study consisted of three treatment groups, Npc1 KO Vehicle, Npc1 KO Cyclodextrin at 4000mg/kg and WT Vehicle; eight, seven and eight animals per group, respectively. All animals were treated starting at D7 (Day 7).
In the literature it is reported that cyclodextrin, once per week given subcutaneously at 4000mg/kg, reverses the NPC disease phenotype in mice. So we set out to replicate those studies using Vium’s novel digital platform. We measured motion, respiration, weight and lifespan. Both motion and respiration were measured with Vium’s cage sensor technology, so the data were constantly being collected and live streamed. Not only were we collecting sensor data, but also video, so we could check in on our mice and observe their movements and behavior at any time, including between 10pm and 12am when I like to send emails, as Vium knows. Body weights were collected one to two times per week and blood was collected at two timepoints during the study, D25 and D59.
We took the study out to 84 days, but of course some animals reached humane endpoint prior to the end of the study. Based on the lifespan metric used by most in the NPC research community as a mouse study endpoint, it was observed that KO Cyclodextrin treated animals live significantly longer than KO Vehicle (Figure 1). As shown below, all KO Vehicle animals die by around D60 while the KO Cyclodextrin animals live beyond D70 and some to the end of the study. One thing to note, is that a few KO Vehicle animals could not tolerate the D59 blood draw and succumbed shortly after. As described in the literature, Npc1-/- animals given the same dose at the same age display a lifespan increase by almost 50%. Our results are on trend with that observed result. Of course we have to take into account factors such as a small n per treatment group, as well as potential variances in severity of disease which may lead to differences in lifespan measurements between our study and what is published. Taking that into account, we believe our results reflect the observed results in the literature.
Figure 1. Cyclodextrin-treated KO animals survive longer than vehicle treated KO controls
Not only do KO Cyclodextrin animals live longer, but they also maintain their body weight longer than the KO Vehicle treated animals (Figure 2). KO Vehicle animals start to show significant weight decline starting around D45, but the cyclodextrin treated animals do not display the same decline in weight. In fact, they maintain weight an average between 15 and 20g post D45. The weight does plateau, and does not continue to increase as seen in WT Vehicle animals, but they do maintain a healthy weight until the end of the study. Weights were measured one to two times per week, one time being prior to dosing with 4000mg/kg cyclodextrin.
Figure 2. Cyclodextrin-treated animals maintain body weight longer than vehicle treated KO controls
In addition to lifespan and weight measurements, we captured novel metrics from the continuous sensor technology in the Vium digital cages, motion and respiration. WT Vehicle animals maintain high levels of activity daily, whereas KO Vehicle treated animals show a decline in motion or activity (Figure 3) around the same time, ~D45, as the decrease in weight is observed (Figure 2). Cyclodextrin-treated animals show a similar increase in motion from D20 to approximately D45 as the other groups, but when the KO Vehicle animals begin to decline, the cyclodextrin-treated animals maintain activity longer, more similar to what is observed with the WT Vehicle animals. Something to note is these data were collected on single housed animals (WT Vehicle n=6, KO Vehicle n=5 and KO Cyclodextrin n=4).
Figure 3. Cyclodextrin-treated animals maintain activity levels similar to activity levels of WT Vehicle animals
For respiration rate, we observed similar results as we did with activity (Figure 4). As mice aged, breathing per minute decreases. WT Vehicle and KO Cyclodextrin animals show decreased breathing rates over time, but follow a similar trajectory to each other, whereas KO Vehicle animals have a steeper rate of decline in respiration rate. This could be due to a number of factors. KO Vehicle mice might take fewer breaths per minute, due to the progression of NPC disease. Or, since the KO Vehicle mice are less active, they might also have a decreased respiration rate as compared to animals that are more active. It is interesting nonetheless and a metric we are looking into using in future studies once we tease apart the aforementioned variables, since it has been shown in the literature that Npc1-/- animal models have pulmonary abnormalities, reflecting what is observed in NPC patients.
Figure 4. Cyclodextrin-treated KO animals and WT Vehicle animals have a similar respiration rate over time
In addition to the metrics mentioned, we collected blood twice during the study. We measured multiple metabolites, but what is shown in Figure 5 and Table 1 are measurements for ALT, AST and cholesterol at D25 and D59, including the standard deviations. As reported in the literature and seen in our results, ALT and AST levels are elevated in KO Vehicle animals as compared to WT Vehicle animals. However, cyclodextrin treatment prevents the ALT and AST levels from becoming elevated to the levels of KO Vehicle animals, and remain more similar to the values from control WT Vehicle animals. This is seen at both timepoints, D25 and D59. In the literature, with this same dosing regimen, it was shown that Npc1-/- mice show improved liver function based on plasma ALT and AST levels.
However, there are conflicting reports as to what happens to cholesterol levels in the plasma post treatment with cyclodextrin. Taylor et al reported that cyclodextrin does not alter the levels of cholesterol in the bloodstream up to 12h post treatment. Our results were similar to that finding although our timepoints of cholesterol measurement varied depending on when animals reached humane endpoint. Another study, conducted by Ramirez et al, determined that cyclodextrin lowered Npc1-/- cholesterol levels in the plasma back down to WT levels. In both of these studies Npc1-/- cholesterol levels were higher than that of WT Vehicle animals.
WT Vehicle, KO Vehicle and KO Cyclodextrin treated animals in our study all have similar plasma cholesterol levels at D25 and D59. Npc1-/- animals do not have higher cholesterol levels on D25 or D59 than the WT levels, and additionally cyclodextrin treatment does not alter those values significantly. Without knowing all of the details from each study, some of the differences between these studies could be due to variability in severity of disease across animals, a small n in each group in our study, differences in mouse strains, or variability in times of collection post cyclodextrin administration.
Figure 5. Cyclodextrin-treated animals maintain reduced ALT and AST enzyme levels as compared to KO Vehicle animals
| Metabolite | WT Vehicle | KO Vehicle | KO Cyclodextrin |
| ALT (U/L) D25 | 53.3 ± 15.8 | 90.2 ± 18.7 | 42.8 ± 3.4 |
| AST (U/L) D25 | 60.3 ± 1.9 | 237.6 ± 27.5 | 90.2 ± 7.5 |
| Cholesterol (mg/dL) D25 | 131.5 ± 4.6 | 121.8 ± 12.4 | 104.4 ± 12.8 |
| ALT (U/L) D59 | 54.5 ± 7.2 | 289.0 ± 100.5 | 109.5 ± 15.5 |
| AST (U/L) D59 | 93/.8 ± 17.8 | 610.3 ± 195.4 | 196.8 ± 30.7 |
| Cholesterol (mg/dL) D59 | 147.8 ± 7.2 | 148.3 ± 17.9 | 128.5 ± 8.0 |
Table 1: ALT, AST and cholesterol levels in plasma of D25 and D59 mice
Again, this pilot study was performed on a small number of animals, but even with the small n, the results are striking. This pilot study run at Vium was successful in reproducing trends in the literature and allowed us to capture new metrics using their digital cage technology. Vium’s technology is ever expending, so while we only collected motion and respiration continuously, there are additional continuous measurements and analyses available since their launch. We look forward to continuing our mouse study work with them in the future. Special thanks and shoutout to Ria Lim, Laura Schaevitz, and the technical staff at Vium who put a lot of time into assisting us with study design and implementation. They continue to help us with developing optimal ways to run mouse studies and capture and analyze established and novel metrics!
