Top 10 Questions: Liver-Targeted Mitochondrial Uncoupling in NHP Models
Dr. Leigh Goedeke, Postdoctoral Fellow at Yale School of Medicine answers the most popular questions from her recent fatty liver disease webinar, covering liver-targeted mitochondrial uncoupling proof of concept studies in NHP models.
1. What’s the Mechanism by Which the Uncoupler is Targeted to the Liver? Are There Effects on Mitochondria in Other Tissues?
CRMP is targeted to the liver by first-pass metabolism after oral delivery. While we have not assessed the tissue distribution of CRMP in NHP models, we have measured the uncoupler in multiple tissues from rodent models of obesity. We found that DNP (the active moiety of CRMP) does not accumulate to levels that increase relative rates of mitochondrial oxidation in the muscle, kidney, heart, or brain.
2. You’ve Previously Shown that CRMP Successfully Increased Hepatic Fat Oxidation and Insulin Sensitivity in Rodents. Which Rodent Models Were Tested?
We’ve shown that CRMP increases hepatic fat oxidation, lowers liver fat, and increases insulin sensitivity in several diet-induced and genetic rodent models of obesity. In particular, we found that CRMP lowers liver fat and increases insulin sensitivity in rats and mice fed a high-fat diet and treated with CRMP for 1-12 weeks. Additionally, we found that CRMP increased insulin sensitivity in ZDF rats treated for 14 days.
We also used a murine model of severe lipodystrophy (fatless A-ZIP/F1 mice) and showed that CRMP treatment reversed hepatic insulin resistance, hepatic inflammation, and diabetes. Lastly, for those interested in NASH, we studied murine models that had been on a methionine/choline-deficient diet for 8 weeks and showed that 6 weeks of CRMP treatment was able to reduce liver fibrosis and inflammation.
3. What is the Effect of CRMP Treatment on Adipose Metabolism and Adipocyte Function/Cell Size/Inflammation?
We evaluated the effect of CRMP on whole-body rates of lipolysis using stable isotope tracer methods in several rodent models of obesity and found no significant difference in insulin-mediated suppression of lipolysis. We have not directly assessed the effect of CRMP on adipocyte cell size and/or inflammation.
4. Why Did the Obese NHPs Selected have Low Hepatic Triglyceride Levels at Baseline?
The NHPs had quite a large age range (9-27 years) and while some NHPs had liver TAGs around 40 mg/g of liver tissue, many did not. Unfortunately, we didn’t screen ahead of time for liver TAGs using liver biopsies and relied on several metabolic parameters to select for dysmetabolic NHPs.
It’s also important to note that the NHPs were on a chow diet for the duration of the study, so it’s possible that as the NHPs aged we would see increased liver TAGs. We are currently working with CrownBio to establish a diet-induced model of NAFLD/NASH in NHPs and plan to test CRMP in this model in the future.
5. Do the NHPs show Histologic Evidence of NASH, Macrosteatosis, Apoptosis (Ballooning), Inflammation, Fibrosis?
We performed histology on the livers from a cohort of the dysmetabolic NHPs and found that most of the NHPs only developed mild steatosis, with little evidence of ballooning, inflammation, and fibrosis.
6. You Showed that CRMP Led to a 30% Reduction in Plasma LDL-Cholesterol Levels. Do You Know the Mechanism by Which this is Occurring?
Previous studies have demonstrated that mitochondrial uncoupling reduces intracellular ATP concentrations, thereby increasing the ADP/ATP ratio and activating AMPK. Consequently, long term CRMP treatment may reduce circulating LDL-C by activating AMPK, phosphorylating and inhibiting HMGCR (the rate-limiting enzyme of cholesterol biosynthesis), and upregulating hepatic expression of the SREBP2-responsive gene, LDLR.
Alternatively, CRMP may activate the SREBP2 pathway by reducing hepatic acetyl-CoA content and cholesterol biosynthesis. Indeed, we found that 6 weeks of CRMP treatment was associated with a 20% reduction in hepatic acetyl-CoA content (as assessed by whole-body βOHB turnover) in dysmetabolic NHPs, suggesting that CRMP may be reducing plasma LDL-C through this mechanism.
7. Mitochondrial Uncouplers have Previously Been Shown to Decrease ROS Production in Many Preclinical Models. Did You See Any Difference in ROS Production in NHPs Treated with CRMP?
We looked at two markers of oxidative stress, the hepatic GSH/GSSH ratio and protein carbonylation. While CRMP treatment did tend to reduce these markers of oxidative stress, it did not reach significance.
8. CRMP Did Not Significantly Alter Body Temperatures - What is the Importance of This Feature?
Mitochondrial uncouplers, such as DNP, shuttle protons across the inner mitochondrial membrane via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production and dissipating the energy as heat. Chronic ingestion of DNP is associated with fatal hyperthermia and was banned by the FDA in the early 1930s due to its low therapeutic index.
CRMP is a controlled-release oral formulation of DNP that is associated with lower peak plasma concentrations of DNP and produces mild hepatic mitochondria uncoupling without causing hyperthermia. Escalating doses of CRMP (1-25 mg/kg) do not increase body temperature in NHPs, which minimizes the toxicity concerns that are inherent to all non-selective mitochondrial uncouplers.
9. Was there any Change in Food Intake?
The NHPs maintained food consumption throughout the study. This is consistent with what we have observed in other efficacy studies performed in NHPs and rodents.
10. Is CRMP going to Clinical Trial, and if so What Will be the Likely Indication?
Our hope is that CRMP will eventually go to clinical trials. Before this occurs several key studies remain. Because of the on-target systemic toxicities associated with mitochondrial uncouplers, future studies using labeled CRMP and whole-body PET-MRI or other methodologies will be important to assess whether DNP accumulates in the brain and/or heart. We are also in the process of planning longer-term safety and crossover efficacy studies in NHPs treated with CRMP.