Merck Double-Knockdown Strategy to Ameliorate Toxicity from Mtp and ApoB Inhibition

The signs are that the next RNAi Therapeutics metabolic/cardiovascular disease candidate will be a dual-targeting one.  While the initial attempts in this area were directed at specifically reducing the well-known cardiovascular risk factor LDL-cholesterol (preferred targets: ApoB and PCSK9), it has become quite tempting to exploit the rare opportunity offered by RNAi Therapeutics to target multiple gene targets with just one formulation to both broaden the therapeutic benefits in patients that typically suffer from a plethora of metabolic dysfunctions (obesity, insulin resistance, and hypercholesterolemia to name a few) and to balance the adverse effects that may result from inhibiting certain targets.

Most notable among the latter is the liver fat accumulation following ApoB knockdown.  Clinical studies with ISIS Pharmaceuticals’ antisense compound mipomersen/KYNAMRO have clearly evidenced such liver fat accumulations which were often accompanied by increases in liver enzymes, general indicators of liver toxicity.  These results further are corroborated by similar clinical observations with the small molecule lomitapide by Aegerion targeting microsomal triglyceride transfer protein (Mtp) which acts essentially at the same stage as ApoB in packaging triglycerides and cholesterol for transport out of liver cells into the circulation.  Aegerion obtained this drug candidate from BMS via UPenn as BMS did not want to further develop this compound due to these safety risks.

Both mipomersen and lomipatide have completed phase III studies and new drug applications for approval in the rare genetic disease homozygous familial hypercholesterolemia (hoFH), and in the case of mipo also for severe heterozygous FH have been submitted to the FDA and EMA.   In terms of therapeutic profile, mipomersen seems to have the edge as, being a phosphorothioate antisense compound, it preferentially accumulates in the liver.  Consequently, it does not cause the side effects resulting from the intestinal inhibition of this pathway that  have been observed with small molecule lomatipe (note: SNALP-delivered RNAi Therapeutics should have similar benefits over small molecules).  Moreover, mipomersen not only lowers LDLc, but also moderately reduces the independent cardiovascular risk factor Lp(a).   Although not a prospective primary goal of mipomersen clinical development, incidental positive findings like this one can go a long way in having regulators take a benevolent look at drug candidates.  This can be seen in the related obesity space where one of the attractive benefits of Arena Pharmaceutical's lorcaserin is that it lowers blood glucose levels.

Obviously, there should be plenty of potential gene targets involved in triglyceride synthesis and utilization/oxidation that could be exploited to concomitantly lower triglyceride content in ApoB/Mtp-inhibited livers while maintaining LDLc-lowering.

Merck Tests ApoB and Mtp Knockdown, Finds Mtp-DGAT Co-Knockdown Promising

Tep and colleagues from Merck published a paper on a study that tested whether an RNAi co-knockdown strategy could be implemented to alleviate the liver fat accumulations due to Mtp and ApoB inhibition.  To be clear, Merck did not state that they have firm intentions of developing such a co-knockdown strategy, but nevertheless noted that such a strategy would have the advantage of  not having to ‘add[ ] a novel compound on top of an approved drug’ and that dual-targeting RNAi Therapeutics candidates are already in clinical development, therefore paving the regulatory path (see ALN-VSP02, and TKM-EBOLA).

In a first step the scientists confirmed the liver fat accumulation following Mtp and ApoB siRNA knockdown.  Not only were they of similar magnitude, the effects of the two knockdowns where essentially the same in almost every other investigated regard.  Notably, there was no reduction in liver fat accumulation following prolonged siRNA treatment as one might have expected based on claims by ISIS Pharmaceuticals of liver fat normalizations with time, but widespread changes in the expression of lipid-related genes were nevertheless observed- this time consistent with claims by ISIS Pharmaceuticals.

Among the genes that were downregulated following Mtp siRNA treatment, presumably as a result of negative feedback, was DGAT2, a key enzyme in triglyceride synthesis that is also thought to represent an important regulatory node in lipid metabolism (e.g. by promoting fatty acid oxidation).  Reasoning that further reducing DGAT2 with liposomally formulated siRNAs may lead to a measurable reduction in liver fat, they then co-formulated the Mtp siRNA with a DGAT2 siRNA and injected them into mice.  Indeed, this resulted in not only the expected LDL-cholesterol reduction, but liver triglyceride increases were mitigated.  According to data not shown, it was claimed that the same beneficial effect could not be observed with an ApoB-DGAT2 siRNA combination, suggesting that Mtp may be the better target for co-knockdown strategies.

It should be added, however, that the day 14 time-point data these conclusions were based on were somewhat of an outlier as at this time the co-formulation with DGAT2 siRNA reduced the LDLc-lowering potency of Mtp knockdown.  On the other hand, the scientists report (also in data not shown) that they tested the co-inhibition strategy using DNA-directed RNAi and thus validated this conclusion.    Moreover, given the multitude of genes involved in lipid metabolism, the Merck scientists stated that the Mtp-DGAT2 co-knockdown is a proof-of-concept and that other targets besides DGAT2 are also being considered (especially Gpat1).

Given Tekmira’s interest in ApoB as a target and recent LNP work by Alnylam and their PCSK9 collaborators from UT Southwestern (Horton, Goldstein) on SCAP knockdown to alleviate hepatic steatosis and Alnylam's general interest in co-knockdown for metabolic applications, Merck will not be alone in their endeavor of finding an RNAi Therapeutic candidate that can do it all, LDLc lowering, triglyceride lowering, and more.  Given that SNALP technology would likely be used in such a clinical program, this could particularly benefit Tekmira.  

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.