Merck Seeks to Optimize Value of ApoB as Target for Hypercholesterolemia with Combinatorial RNAi Therapeutics

A series of recent papers (e.g. Ason et al. 1; Tadin-Strapps et al.; Ason et al. 2) shows that Merck wishes to use RNAi Therapeutics for the treatment of hypercholesterolemia, a precursor of cardiovascular disease. Despite the success that widely prescribed drugs such as statins have had in lowering bad cholesterol, there are still many patients in need of additional treatment options, patients that either have very high cholesterol levels to start off with or those that do not respond to or tolerate these drugs. As a result, interest in the pharmaceutical industry remains high in developing new approaches to treat these underserved patient populations.

The Pros and Cons of ApoB as a Target

ApoB has emerged as a very attractive, hitherto undruggable target in this endeavor, and indeed ISIS in partnership with Sanofi-Aventis are currently knocking on regulators’ doors to get their ApoB-targeting RNaseH-antisense drug mipomersen (commercial name: Kynamro) approved for familial hypercholesterolemia. Meanwhile, Tekmira and apparently Merck are in the early stages of developing ApoB-based RNAi Therapeutics for hypercholesterolemia.

What makes ApoB such an attractive target is that it is the critical protein component of bad LDL cholesterol (LDLc) and knocking it down therefore very potently reduces LDLc levels in the blood. Not only that, it seems that through the wonders of sophisticated feedback control mechanisms of lipid metabolism, essentially all other atherogenic lipids are reduced, too, following ApoB knockdown (e.g. Tadin-Strapps). There remain, however, two concerns with ApoB as a target.

The first one relates to the observation that in most rodent models, not only all the atherogenic lipids are reduced, but also the ‘good’ HDL-cholesterol which is responsible for reverse cholesterol transport from the plaques (where they are dangerous) back to the liver for excretion in bile (which is where they belong to). Research by Merck, of course using LNP technology, shows that when ApoB is knocked down by ~95%, both HDLc and LDLc where reduced by more than 2/3 (Tadin-Strapps et al. 2011; 79-90% non-HDLc lowering and 67-78% HDLc lowering in Ason et al). This was highly unlikely due to an off-target effect as various ApoB-targeting siRNAs exhibited this phenotype while non-targeting LNP formulations did not.

Nevertheless, it is unclear whether these rodent and similar non-human primate findings translate into humans, and what ApoB knockdown levels would need to be achieved to start seeing an effect on HDL. Mipomersen e.g. reduces LDLc by about a third and does not seem to affect HDLc in humans. The Merck scientists also speculate that the HDLc reduction simply reflects that in the absence of LDLc, ApoE redistributes to HDLc leading to their more rapid uptake in the liver. Therefore, despite the mantra that it is all about the HDL:LDL ratio, HDLc reductions via this route would actually be positive.

The second, probably more pressing concern is that ApoB inhibition leads to a failure to export lipids from and their accumulation in the liver, a condition known as hepatosteatosis or ‘fatty liver’. This has not only been observed in pre-clinical studies of ApoB knockdown, but was also observed in the mipomersen clinical studies (Visser et al. 2010). ISIS Pharmaceuticals, the discoverers behind mipomersen, argue that this accumulation is likely to be temporary only as compensatory genetic circuits get switched on to reverse the phenotype, a mechanism that is supported by Merck's own gene expression analysis. Moreover, it has yet to be shown that ApoB-related fatty liver predisposes to the development of liver fibrosis and ultimately liver failure or cancer which is really why we care about fatty liver in the first place.

Enhancing the Therapeutic Profile of ApoB-targeting Drugs

Giving up on ApoB in hypercholesterolemia because of the fatty liver concerns would mean forfeiting the potential of one of the most if not the most potent target in the hypercholesterolemia space. I therefore fully agree with the strategy by Merck to exploit the combinatorial potential of RNAi Therapeutics to optimize the profile of an ApoB-targeting RNAi Therapeutic, a strategy that I would fully expect of Tekmira to be evaluating as well.

The combinatorial potential of RNAi Therapeutics is one of the major attractions of this technology. Because of the almost identical pharmacological behaviors of siRNAs, it is relatively simple to employ multiple instead of just a single siRNA payload in an RNAi Therapeutic. This is particularly useful for complex diseases such as metabolic syndromes and diseases that involve resistance/escape such as cancer and viral infections. ALN-VSP02 is a dual-targeting example in oncology that is already in the clinic, and Tekmira’s Ebola RNAi Therapeutic candidate slated to enter the clinic in early 2012 will also involve at least two different RNAi triggers.

You can thus imagine that knocking down a gene along ApoB that leads to increased lipid excretion via the bile, increased fat oxidation in the liver, or redu­ced hepatic fat synthesis or reduced uptake of dietary fats in the liver, would greatly enhance the therapeutic profile of an ApoB-based drug by countering the development of fatty liver. It is the latter approach that Ason and colleagues from Merck took in their recent paper by targeting fatty acid transport protein 5 (Fatp5) alongside ApoB as Fatp5 had been described, also through the elegant application of ddRNAi, to reverse diet-induced hepatosteatosis.­

To study the effect of Fatp5 co-knockdown on ApoB-induced fatty liver, the researchers formulated both siRNAs into LNPs and infused them into mice. Both genes were knocked down efficiently (89-95%) and as you can imagine, at these ApoB knockdown levels, the fatty liver phenotype was quite robust. Predicted Fatp5-dependent phenotypes, such as an almost 1000-fold increase in the ratio of unconjugated to conjugated bile acids in the bile, were also observed (Fatp5 plays a role in bile acid conjugation) confirming the functional knockdown of both ApoB and Fatp5.

Unfortunately, despite the potent knockdown of Fatp5, the ApoB-dependent fatty liver phenotype was not reversed in the mice which were fed a ‘Western low-fat diet’. It therefore appears that Fatp5 intervention is only useful for diet-induced fatty liver, and that approaches specific to fat excretion or fatty acid oxidation in the liver will be more promising. Nevertheless, the scientists seem to be on the right track, and with SNALP siRNA delivery, it should be relatively easy to characterize other candidate genes. Indeed, due to competition, the Mercks, Tekmiras, and Alnylams may not necessarily want to disclose their magic siRNA cocktail.

So as we are on the eve of seeing mipomersen being approved as the first ApoB-targeting compound for the treatment of hypercholesterolemia, a second generation of ApoB-targeting RNAi Therapeutics are being readied that not only aim at incremental improvements in potency and dosing frequency, but completely rehaul the therapeutic profile of ApoB-based therapeutics.