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.  

An Exemplary RNAi Therapeutics Target

A paper by Alnylam and collaborators at Harvard and the MIT on a strategy to treat anemia recently appeared in Blood and involves an exemplary RNAi Therapeutics target gene selection which takes into account both the scientific and technical strengths and challenges of the technology (Querbes et al. 2012).  In addition, it is yet another demonstration of the powers of Tekmira’s SNALP technology and the various therapeutic opportunities that it can address with gene knockdown in the liver alone. 

Before going into the narrative of the paper and risk losing your attention, here are the main reasons why the target gene(s), PHD1-3, uniquely lend themselves to an RNAi Therapeutics approach (as opposed to competitive small molecules and recombinant proteins/MAbs) and why SNALP delivery is a particularly good match for them:

1. The target genes (PHD enzymes) are cell-autonomous negative regulators of the expression of a secreted factor (here: erythropoietin/Epo).  This means that even if efficient RNAi knockdown were only achieved in a subset of cells, the secreted factor that will be generated in this subset of cells can still act globally through the systemic circulation.  This is in contrast to other gene targets, such as certain intracellular targets involved in cancer cell proliferation, where efficient gene knockdown would be required in almost every cell to achieve a measurable outcome. 

2. Specific multi-targeting. In order to exploit a genetically intriguing mechanism for the treatment of disease (PHD regulation for the treatment of anemia), it was necessary to simultaneously knock down three gene family members.  Small molecule inhibitors with such multi-targeting activity would most likely cross-react with additional family members, increasing the risk of (A) obtaining confounding biological outcomes as other family members are more likely to play roles in related biological pathways than e.g. a spurious RNAi off-target event that is pathway-independent, and, of course, of (B) off-target toxicity.  The next time you hear about that exciting multi-targeting kinase inhibitor approach in oncology, remember that many of these were not multi-targeting by design and that there will be a number of additional kinases being hit that you will hardly hear about.  In summary, it is not just the capacity of RNAi Therapeutics for multi-targeting (e.g. as in the clinical candidates ALN-VSP02, TKM-EBOLA), but it is also the more ‘wholesome’ multi-targeting mechanism that is an advantage of the technology here. 

3. Tissue-specific delivery (here: SNALP and liver). Non-specific, but also specific targeting when it occurs in the wrong tissues, may cause toxicities.  In this example, because the gene family to which the target genes (PHD enzymes) belong play roles in various non-targeted biological processes throughout the body, there is great concern that a small molecule PHD inhibitor would cause a number of toxicities.  With regard to on-target toxicity in the wrong tissues, the HIF transcription factor that is regulated by the PHD1-3 enzymes plays an important role in cancer biology and inhibiting it all over the body may elevate the risk of developing cancer.  On the other hand, with the SNALP-formulated PHD siRNAs most of the knockdown can be directed at the desired liver so that the numerous on- and off-target toxicities in the rest of the body become irrelevant. 

4. Being an exclusively intracellular target means that it is not druggable by monoclonal antibodies = less competition. 

5. Recombinant protein therapy (e.g. rhEpo) often involves periods of supraphysiologic exposures which can be associated with adverse events (this is thought to be one problem with rhEpo therapy).  By contrast, (genetic) PHD knockdown allows for more physiologic regulation of the targeted biological pathway making it a potentially safer and also more efficacious therapy.  This advantage of affecting a more physiologic pathway compared to the competing recombinant protein approach is largely the result of having a larger target space to choose from.

Summary of paper

The motivation for the study by Querbes and colleagues is the fascinating insight that even in adulthood the liver continues to be a potential source of physiologically relevant levels of erythropoietin (Epo), a key red blood cell-stimulating factor that is almost exclusively produced by the kidneys in adults under normal conditions.  Nevertheless, it is the liver that is the site of Epo production until around birth when erythropoiesis switches from the liver to the bone marrow. At that point, hepatic Epo transcription is suppressed by the degradation of the important Epo transcription factor HIF following the activity of PHD enzymes 1, 2, and 3.  When these enzymes are removed, e.g. by genetic excision, the liver can produce similar levels of Epo as the kidneys even in adult mammals.

Clearly, constitutive genetic excision is not an option when it is important to achieve just the right level of a physiologic process (here: red blood cell volume).  The anemia drug development field in particular has been tarnished by companies and physicians having excessively pushed the use of high-dose recombinant Epo (rhEpo) and other erythropoiesis-stimulating agents (ESA).  In some patient groups, this has been shown to greatly increase the risk of thrombotic events and other side effects that may be unique to ESAs.

Oral small molecule inhibitors of HIF-related PHDs (HIF-PHIs) have been tested in clinicaltrials by Fibrogen for the treatment of anemia in end-stage renal disease along a more conventional treatment paradigm.  Although the clinical experience so far suggests a good safety profile, until larger patient numbers have been exposed for prolonged periods of time, there will always be concern about toxicities from the inhibition of on-target and off-target PHDs, especially in non-liver, non-kidney tissues.

By contrast, the SNALP-siRNA approach taken by Querbes and colleagues targets PHD1-3 almost exclusively in the liver by virtue of  short PEG-anchored SNALP* biodistribution.  Moreover, the nature of siRNA selection and off-targeting makes it much less likely that confounding or adverse off-targeting will be encountered.  

70-80% target gene knockdowns were achieved for PHD 1 and 2 and a somewhat less pronounced ~50% knockdown for PHD3 (probably due to a previously observed feedback mechanism) when measured across the liver in rodents.  Despite the incomplete overall knockdown, this resulted not only in several log-fold increases of liver Epo mRNA, but also pronounced, highly physiologically relevant levels of serum Epo.  These responses can be likely attributed to the RNAi Therapeutic candidate targeting negative regulators of gene expression.

Commercial potential

The anemia market has long been dominated by rhEpo and related protein-based ESAs.  Only recently, a peptide-based ESA by Affymax (Omontys/Peginesitide) was approved by the FDA as a new treatment option and should help bring down costs.

Although all these ESAs do a good job in elevating hemoglobin content, the opportunity for new agents is in doing just that, but with a better safety profile as all currently approved ESAs suffer from the above-described concerns arising from supraphysiologic exposures.  Thus, the hope with the new anemia drug candidates is that the targeting of alternative biologic pathways won’t suffer from the same limitations.  They may also increase the quality of the hemoglobin elevation, especially with regards to iron metabolism.

Despite the promising data, it is as yet unclear whether Alnylam will develop PHD inhibition to treat anemia.  Although refractory anemia is among the 5x15^TM programs, Alnylam hasindicated that it is more interested in modulating the hepcidin pathway for that indication (hepcidin affects erythropoiesis via iron metabolism).  I’m not sure though whether the choice is not partly 2due to the fact that Fibrogen seems to have a respectable IP position in PHD inhibition.  Incidentally, the corresponding author of the paper, Prof. William Kaelin from Harvard, has a financial interest in Fibrogen.  Also of note, some of the transgenic mice used in the study originated with Regeneron.

*Alnylam tight-lipped about specific SNALP formulation used in study

It is not surprising that, like all of Alnylam’s other commercially interesting applications, also the anemia program is based on Tekmira’s SNALP technology.  Because of the litigation between the companies, it is of interest that while the methods section described much of the SNALP formulation, it conspicuously failed to mention the identity of the ionizable lipid, although based on the observed potency it should be a ‘second’ or ‘third’ gen lipid:

‘siRNA Formulation in Lipid Nanoparticles

The LNPs were prepared with an ionizable lipid, disteroylphosphatidyl choline,

cholesterol, and PEG-DMG using a spontaneous vesicle formation procedure as

previously described at a component molar ratio of ~50/10/38.5/1.5 Ref 17,18.’ 

Ref 17: Akinc A, Querbes W, De S, et al. Targeted delivery of RNAi therapeutics with

endogenous and exogenous ligand-based mechanisms. Mol Ther. 2010;18(7):1357-1364. Study on the ApoE-mediated mechanism of SNALP delivery to hepatocytes (Tekmira claims this was their trade secret which Alnylam chose to publish and claim to be its own insight)

Ref 18: 18. Semple SC, Akinc A, Chen J, et al. Rational design of cationic lipids for siRNA delivery. Nat Biotechnol. 2010;28(2):172-176. Rational SNALP design paper by Tekmira (the contested ionizable MC3 lipid was derived from the rational design approach among others)

Alnylam said that it was planning on partnering the anemia program for phase I studies.  By disclosing their intention to partner at such an early stage, it almost seems like they have been in advanced talks with potential partners.  Otherwise, promising partners at such an early stage would only set the company up to disappoint.

I would therefore assume that Tekmira will be looking at this paper for evidence for whether, also in this case, Alnylam inappropriately shared SNALP reagents and insights with ‘3^rd parties’.  As a reminder, according to the Manufacturing Agreement between Tekmira and Alnylam, Tekmira is the sole SNALP supplier, also for pre-clinical research purposes, and Alnylam is prohibited from providing ‘third parties’ with such formulations (and insights).  

‘Third parties’ ought to include academic collaborators such as Prof. Kaelin and the other researchers from Harvard and MIT on this paper.  It is interesting to speculate that this was a reason for the secrecy around the ‘ionizable lipid’?     

ASCO for RNAi Therapeutics in Line with Expectations, but Curious Nevertheless (Part II- Atu027)

With some delay, only mirroring the strange delay by the sponsor company, Silence Therapeutics, in disseminating the results, here are my thoughts on the Atu027 phase I results presented at this year's ASCO and some speculations on the potential corporate fate of Silence Therapeutics.  

For part I (ALN-VSP02 ASCO discussion) here.

AtuPLEX Delivery Tech Tolerated Up to Twice Dose Required for Endothelial Knockdown

In retrospect, the phase I Atu027 surprised to the upside, especially in that the relatively ‘unsophisticated’ (some would say simple = elegant) AtuPLEX formulation used was apparently well tolerated in humans at doses well above where we would expect target gene knockdown in endothelial cells, the target cell population of this RNAi delivery technology: the 0.18mg/kg in this study yielded plasma siRNA concentrations where noticeable knockdown was seen in preclinical monkey studies in endothelial cells, and 0.336mg/kg is the recommended dose for further studies.   

I should caution, however, that the PK data are strictly inferring endothelial cell knockdown in the lung (of monkeys) to knockdown in tumor endothelia.  Although the data indicate that AtuPLEX (unlike e.g. DACC for lung endothelia) has a fairly broad target spectrum of endothelia in various tissues, there are some slight differences.  

On the downside, this study in patients with advanced solid tumors failed to provide striking evidence that Atu027 has indeed anti-tumor efficacy.  Although not a primary goal of this dose-finding dose escalation study, with only a paucity of efficacy-related data collected in this trial (in stark contrast to Alnylam’s heroic efforts with ALN-VSP02), it would have been comforting to see more evidence of efficacy besides the two reported regressions of a lung and a liver met plus the ‘stable disease responses’ -which really mean little in the absence of a control group.  The biomarker data were certainly curious, but without disclosing the full dataset could have been as well a cherry-picking exercise.  On this note, I also would have liked for Silence Therapeutics to disclose the full PK dataset.  

Besides for the small patient numbers (33), an explanation for a possible failure to see anti-tumor efficacy could be the choice of target gene, PKN3 (downstream of PI3K), which is a new clinical molecular target in the oncology arena.  On the other hand, I am pleased to see others in the blogosphere point out that the choice of a higher-risk target may be more than compensated for by the differentiation value it brings.  So in this case, you not only have an RNAi mechanism of action, but also the drug target as two major value-adding differentiating factors (see the March of the Lemmings by Bruce Booth).  But if target choice eventually turned out to be a problem here, the Atu027 PK results represent an important de-risking for the AtuPLEX delivery platform.   

Financial worries to the fore 

I’m sure many will be happy that this trial has finally wrapped up (last patient dosing expected later this month).  However, this also means that the focus will now be on a much-feared financing; feared, because the last financings by Silence Therapeutics have been catastrophic to existing shareholders. It is a shame that Silence Therapeutics has failed to obtain any non-dilutive funding despite numerous opportunities.

Along with the disclosure of the ASCO presentation, the company thus announced in an quite unusual move that ‘advanced discussions’ were under way to raise 4-5M UK pounds.  Judging from the previous fund-raisings, this may well double the share count.  Despite such a dilutive capital raise, the terms for which would more likely than not be dictated by the new investors, it would still be preferable over a Marina Bio-style cash crunch.  Actually, from where I sit in my armchair, a combination between the two ‘second-tier’ players Silence Therapeutics and Marina Biotech does not look that illogical as long as can find ways to further cut down on cash burn.  Note that some of Marina Biotech’s important business relationships are with European companies: Debiopharm, Girindus, Novosom tech and IP, usiRNA IP from Denmark etc etc.  

I'll be watching intently as to what the next move will be...

ASCO for RNAi Therapeutics in Line with Expectations, but Curious Nevertheless (Part I- ALN-VSP02)

Alnylam and, finally, Silence Therapeutics have by now disclosed the latest results from the phase I studies of their RNAi Therapeutics candidates, ALN-VSP02 and Atu027, for solid cancers at this year's ASCO.  As the studies have thus more or less come to a conclusion, one has to say that, although clearly nothing spectacularly positive came out of them, these two candidates warrant further evaluation and their safety and pharmacokinetic profiles augur well for the other candidates based on the same SNALP and AtuPLEX delivery platforms.  

With most of the results known, I will therefore focus my discussions of the RNAi Therapeutics ASCO 2012 on the new insights and include some comments on recent corporate maneuvers of the companies involved.

  

ALN-VSP02 Provides First Insights into SNALP Repeat Dosing (and yes, SNALP technology belongs to Tekmira and AlCana is an instrument of Alnylam)

The extension study with ALN-VSP02 that was the subject of Alnylam's ASCO poster presentation included 7 patients with cancers involving the liver that had been rolled over from the main study (results presented a year ago here) and provided more insights into the long-term safety/tolerability of SNALP delivery technology.  It was good to see that even very sick patients could be given 1^st generation SNALP formulations at dosages of 0.7mg/kg or more every other week for quite prolonged periods of time (up to 2 years).  As the phase I results with ALN-PCS02 indicate, such concentrations will allow for solid gene knockdowns in normal liver cells with some of the improved SNALP formulations. When I first got excited about SNALP delivery technology 6 years ago, one of the main risks that I saw was around repeat-dosing including antibody formation against the PEG component and other immune stimulations upon repeat dosing.  On that front at least, the ALN-VSP02 study, especially based on the PK data, was a clear pass. 

Certainly, there remains controversy around whether the precautionary transient pre-treatment with immunosuppressives (e.g. corticosteroids) that was applied here and may have aided in achieving these results is a realistic product feature of a RNAi Therapeutics.  As I have said before, personally I believe that for severe indications such as many cancers, this is mostly a minor practical inconvenience; it may, however, impede their adoption for less severe conditions in larger patient populations.

One new safety-related item worth mentioning is the reported effect of prolonged ALN-VSP02 administration on spleen size and Alnylam’s interpretation thereof.  As we know, the spleen is a non-essential organ in humans and the vast majority of us would not notice if we were without one.  Thus, the reduction in spleen size observed in this trial with prolonged treatment was not considered a severe adverse event or dose-limiting toxicity.  Spleen toxicity would not really be surprising for nanoparticle-formulated drugs such as ALN-VSP02 (and also phosphorothioate-based oligonucleotide therapeutics) as they are often observed in animal studies.  I therefore considered it possible that it could be a platform-based toxicity.  

Alnylam, however, put forward a more optimistic interpretation by claiming that it is more likely an on-target toxicity, meaning that knockdown of the proliferative gene KSP in the spleen was responsible for it.  Considering the biodistribution of SNALP and the recently highlighted data on SNALP and other LNP-mediated gene knockdown in immune cells that are enriched in lymphoid organs such as the spleen, this is certainly a plausible explanation as well and is further supported by the new monkey data presented at ASCO.  Adding to this confusion is the fact that ‘Alnylam’ has been developing 3^rd generation SNALP technology to ameliorate spleen toxicity.  So if the spleen shrinkage was no general effect, why bother with 3^rd generation?

Aside from these scientific developments, including a very nice complete response by RECIST in an endometrial cancer patient with multiple liver mets which had already started to respond during the main phase of the trial, my interest was equally piqued by the fact that the press release by Alnylam lacked any mention of the fact that ALN-VSP02 involves critical delivery technology by Tekmira and that key IP of a siRNA ingredient may also well belong to Tekmira, pending the outcome of an Interference proceeding in the US.  If you look for even more support for Tekmira’scontention that Alnylam has been aiming to marginalize the company, and as yet another of Alnylam's main rivals, Marina Biotech, looks like it is going out of business (my bet is that they will participate in some kind of consolidation to survive in another form), look no further.  The PR is only rivaled in silliness by the misleading one issued by Alnylam’s Canadian subsidiary, Alnylam Canada (AlCana), two weeks ago on the status of the Tekmira-Alnylam showdown in Canada (the misleading part being in quite obviously trying to suggest that the Canadian courts reversed all earlier decisions that went against AlCana, including handing over the allegedly stolen documents back to their rightful owners, Tekmira). 

But the real clincher of that PR was the following: despite of pointing out the elephant in the room, namely that the Canadian proceedings are part of a wider confrontation between Tekmira and Alnylam, Alnylam Canada hilariously and awkwardly at the same time failed to mention Alnylam at all- in the entire press release!  If they believed that this would make AlCana, a de facto academic lab financially supported and strategically instructed by Alnylam all along, any more independent of Alnylam, try again (note: a key element in the litigation is that Alnylam established an ‘independent’ company- AlCana- to gain insights into key Tekmira trade secrets and know-how thereby minimizing legal liability if caught).  More unintended humor was provided by stating that the Canadian case had been post-poned until resolution of the US case between the ‘corporate’ parties, implying that neither AlCana nor the courts consider AlCana to be a true, independent corporation.  Although I have not had the chance to read the primary court documents myself, the decision strongly suggests that the Canadian court took pity on the AlCana scientists and does not like to see them being thrown under the bus by their de facto employer, Alnylam.

In part 2, I will cover the latest data on Atu027 and share some thoughts on the confusing corporate strategies of that company.