Alnylam Indicates that 80% Subcutaneous TTR Knockdown Achievable with Magic 2.5mg/kg Dose

This morning, Alnylam announced a milestone in the development of subcutaneously administered, systemically acting RNAi Therapeutics.  A ‘more than 80%’ knockdown was achieved in a phase I trial of ALN-TTRsc, the GalNAc-siRNA conjugate for the treatment of TTR amyloidosis.  It is the first time that a meaningful knockdown in Man was reported using the subcutaneous route of administration.

The key question as to the clinical attractiveness of the liver-specific GalNAc delivery platform versus intravenous alternatives such as SNALP (compare to just reported ALN-TTR02 clinical results) relates to injection volume and frequency of administration.  The hurdle to overcome here would seem a once weekly regimen with dosages of 2.5mg/kg or less.  2.5mg/kg is widely considered to be a threshold as this amount of drug can be accommodated in a 1ml syringe.  Higher injection volumes would likely be quite painful and possibly increase the risk of injection site reactions.  Higher dosing frequencies in order to stay within the 2.5mg/kg limitations would reduce the convenience factor.

Unfortunately, these data points were left undisclosed in today’s announcement.  Instead, we were given a riddle.  The company first stated that the results were in-line with observations in non-human primates.  At another point, they say that 80% knockdown with ALN-TTRsc were achieved in non-human primates with the magic 2.5mg/kg dose.  Logically, Alnylam stated that in humans 80%’ knockdowns were seen with the 2.5mg/kg dose. 

Curiously, the company also stated that dose escalation was continuing.  It makes, of course, sense to push the degree of knockdown.  The difference between an 70% knockdown (e.g. ISIS-TTRRx), an 80% knockdown (current ALN-TTRsc results), and 90% knockdowns (e.g. ALN-TTR02) is that compared to a 90% knockdown, the amount of remaining insulting protein (here: mutated TTR) is twice (80%) and 3x (70%) as high which could result in dramatic differences in the clinical outcome, or even whether a knockdown approach would even work for a disease like TTR amyloidosis.

Maybe we will get more clarity on dose and dose frequency at today’s R&D day.  If not, more detailed results are to be presented at the Annual Scientific Meeting of the Heart Failure Society of America (HFSA), September 22-25.

RNAi Therapeutics Ebola Candidate Achieves Critical Milestone

Yesterday at the TIDES meeting, Tekmira reported new efficacy data for its Ebola RNAi Therapeutics development program.  The non-human primate data suggest that the main remaining hurdle before regulatory approval for this biodefense agent (on which any government stockpiling contract would hinge), safety in healthy human volunteer studies, is now more realistic than ever.     

Is second time's the charm?

In 2010, Tekmira and collaborators from the Geisbert lab and the US Army reported in The Lancet of a breakthrough in the post-exposure treatment of Ebola infection.  In the gold-standard rhesus monkey model for Ebola infection, their were able to demonstrate that SNALP-RNAi could effectively rescue Ebola-infected animals from near-certain death. 

The efficacy, however, depended on doses of 2.0mg/kg is likely too high a SNALP dose to be considered safe.  Based on the SNALP human clinical trial experience to date, first dose-limiting toxicities are observed at 1.0mg/kg (note: SNALP potencies are very comparable between humans and monkeys).  Moreover, at doses higher than 0.5mg/kg, sporadic signs of immune stimulations (flu-like symptoms) can be observed.  These caused Alnylam and Tekmira to adopt precautionary transient immune suppressions with corticosteroids in some of their other SNALP-based clinical studies.   

It therefore became important to take advantage of the improvements in SNALP potencies and develop more potent Ebola SNALP-RNAi candidates.  Fortunately, Tekmira was able to get the blessing from the US Department of Defense for this, and announced in April that it would receive up to ~$7M in additional funding on top of the up to $34M initially agreed upon in order to bring such a candidate to the end of phase I clinical studies. 

Importantly, since Ebola efficacy trials cannot be conducted in humans, the regulatory approval of TKM-Ebola will be according to the Animal Rule.  The Animal Rule states that for indications where human efficacy trials are not possible due to ethical considerations, it is sufficient to show efficacy in accepted animal models and then establish drug safety at corresponding doses in healthy human volunteer studies.  

With the new formulation, 12 out of 12 monkeys treated with the magic dose of 0.5mg/kg RNAi stayed alive in a post-exposure treatment setting, compared to only 1 out of 6 treated with placebo.  This means that should the human volunteer trials show good safety at the same 0.5mg/kg dose level, Tekmira will stand a good chance at gaining regulatory approval under the Animal Rule.  Of note, the first Ebola trial were likely conducted in the absence of corticosteroid pre-treatment and it is very likely, also in light of Alnylam's recent announcement of tapering off corticosteroids in the ALN-TTR02 trial, that the future TKM-EBOLA studies will also be conducted without them.

Since 0.5mg/kg is right on the border, however, there still remains an element of risk before victory can be declared on the safety front.  It is therefore good to hear that 4 out of 6 monkeys stayed alive at the 0.2mg/kg dose. This should provide a good safety cushion despite the aggressive daily treatments that will be required in this demanding setting.

Critical human safety data, ideally from repeat-dosing studies, should be forthcoming in 2014 as Tekmira has guided that it will commence clinical studies with the new candidate by the end of 2013.

Update 16/5/2013: One issue on the efficacy side of the equation that I forgot to mention in the original post is that the DoD might ask the company to test TKM-EBOLA with longer delays after the infection in monkeys.

SNALP Shows Good Potency with Subcutaneous Dosing

Also at the TIDES meeting, Tekmira presented efficacy data for SNALP when given via the subcutaneous route of administration.  To wit, SNALP has largely been administered intravenously which may not be suitable for a number of indications, especially the lower risk, chronic diseases.  Pfizer once famously criticized the state of RNAi delivery as being limited to intravenous options.

Comments like these (which are very relevant for partnering purposes) and trends in the industry to adopt subQ dosing, most notably Arrowhead’s DPCs and Alnylam’s GalNAc-conjugates, probably motivated Tekmira to explore this route of administration with SNALP further.

 

Based on an early ApoB patent application, Tekmira had clearly had some familiarity with subQ, and I had always wondered why the company did not pursue this further.  It could be that the more potent SNALP chemistries now allow for much more potent and robust subQ RNAi gene knockdown.

The new data show a very impressive 96% knockdown of transthyretin (TTR) with a single subQ administration of 1.0mg/kg SNALP in rodents.  This compares to a much more modest ~55% knockdown with ALN-TTRsc at 1.0mg/kg given on 5 consecutive days, the subQ GalNAc-siRNA candidate by Alnylam which just entered clinical development for the FAC form of transthyretin amyloidosis.

Of course, we need to learn more about the safety of subQ SNALP to know whether it really is a clinically viable strategy and superior to the competition.  GalNAc-siRNAs in particular look quite benign, albeit not very potent.  However, since peak plasma concentrations seem limiting for SNALP in terms of safety (infusion reactions can be managed by slowing the infusion), subQ, due to its slower systemic release might actually turn out to be safer, not just more convenient.  On the other hand, my impression is that subQ SNALP could cause the type of ‘nuisance side effect’ that is frequently seen with phosphorothioate antisense.

The GalNAc comparison is obvious since Alnylam developings both a SNALP- and a GalNAc-based TTR RNAi Therapeutics in parallel.  In terms of competition for potential partnering, however, the DPCs by Arrowhead Research would be the more appropriate and challenging comparison.  I look forward to watching the race unfold as healthy competition is propelling the RNAi Therapeutics field into a highly competitive industry.

Is Alnylam Foolishly Betting the Farm on GalNAcs?

It is remarkable that Alnylam seems to be embracing GalNAc subcutaneous delivery for all pipeline candidates, but for perhaps ALN-TTR02, that are most important to its 5x15^TM development and commercialization strategy: ALN-TTRsc (TTR amyloidosis), ALN-AT3 (hemophilia), and ALN-AS1 (acute intermittent porphyria).  Considering that gene knockdown has not been demonstrated in Man with GalNAc conjugates, and potency could be a critical issue determining whether this really is a subQ approach, this apparent high-risk strategy is uncharacteristic of a company with the laudable attitude of raising capital when the company does not need it.   

ALN-TTRsc: The Missing Clinical Trial

One possible reflection of the fact that management does not really feel as confident about the strategy as it may seem, is the missing entry of the ALN-TTRsc phase I clinical trial on clinicialtrials.gov (and other registries I looked at).

I like to look at clinical trial registries, esp. clinicaltrials.gov, also for the reason that information contained therein often provide interesting clues into otherwise undisclosed safety and efficacy issues.  For example, is it a single and/or multiple dose, what are the dosing ranges, etc. 

With ALN-TTRsc, I do not expect safety to be necessarily the dose limiting factor.  Rather, it may well be the ability to show robust efficacy with an injection volume of 1ml or less, the magic upper limit for subQ approaches.  It turns out that adequate efficacy was seen in non-human primates only starting at 2.5mg/kg which apparently corresponds to the 1ml volume in humans.  Moreover, the dose-response curve is relatively shallow making it difficult to extrapolate predicted doses from preclinical models into humans (being off by a factor of 2 in the ED50 could already prove disastrous).

Is the omission to list ALN-TTRsc on clinicaltrials.gov an attempt at hiding that the dose escalation schedule indicates that 2.5mg/kg is an optimistic guess? Of course, the secrecy could also be for competitive reasons in light of its race with the competing antisense approach by GSK-ISIS.  But for a company that so far has listed every clinical trial, including ALN-TTR01 and ALN-TTR02 all of which have been conducted exclusively outside the US, there will be a reason for it and doubts about GalNAc potency is a plausible one.

GalNAc Steals Spotlight from DPCs

Recently, Arrowhead scientists presented impressive subQ hepatic knockdown data with the DPC polyconjugate platform.  While the response to the presentation at the Oligonucleotide Society Meeting in Boston last year was tremendous, possibly the most enthusiastic one among all the presentations (and one of the reasons why I am bullish that there is wider industry demand for DPCs), Arrowhead has been getting no love from the financial markets, thus putting it at a disadvantage in any licensing negotiations.  One factor for that might be GalNAcs which take away in the typical investor’s mind the uniqueness about it being a subQ RNAi delivery approach.  So if Alnylam eventually realizes that it needs an alternative, it might get access to it on more affordable terms.  The phase I ALN-TTRsc results expected mid-year could be the critical event in that dynamic: poor results would benefit Arrowhead Research.

GalNAc Plus

Of course, the confidence in GalNAcs may rest in the knowledge that ALN-TTRsc is actually the pipeline candidate with the weakest potency, and if IT shows efficacy, it will be a stroll for all the others.  These apparently have proven to be more potent in preclinical studies.  

I consider this a possibility, but am not fully convinced that these conjugates can really be called GalNAc-siRNA conjugates.  This is because the TTR siRNA is a particularly potent one already and I am wondering how you would achieve a 10x increase in potency from ALN-TTRsc to ALN-PCSsc with stabilizing nucleic acid chemistry alone.  Moreover, it is a small miracle already that a simple siRNA with a targeting ligand can achieve ED50s in the low mg/kg, because the related cytoplasmic release would likely rely on spontaneous endosome rupture (miracle probably explained by the high volume ASGPR receptor-mediated trafficking).

Consequently, my feeling is that the GalNAc Plus conjugates involve additional functional moieties beyond stabilizing chemistries such as 2'-o-methyl and 2'-F…such as endosomolytic activities.  In a 2009 patent application by Manoharan et al. (WO 2009/126933) such endosomal release activities, melittin included, were indeed contemplated in the context of GalNAc-siRNAs.  In fact, the claims would cover GalNAc-targeted DPCs such as the one described in the Mirus/Arrowhead in the 2007 PNAS paper (Mirus cientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes).

The patent application, somewhat of an attempt at an early land-grap being devoid of any actual experimental data, is not very remarkable really as it is obvious to somebody skilled in the art that in order to significantly enhance GalNAc-siRNAs such functionalities will be highly desirable, so a lot of it would come down again to chemistry and figuring out which exact compositions are functional and safe.

In my mind, it is fair game to pursue promising (delivery) technologies, especially under the Research Exemption in the US.  However, as the Tekmira-Alnylam relationship has shown, it is foolish for two companies to collaborate on delivery when both are working on GalNAc conjugates internally.  At best, the technology is useless and the whole thing will be forgotten.  At worst, the technology is valuable and both parties will try to control it and not pay the other party their dues.   

Overall, I think that betting the farm on GalNAcs is taking on more risk than warranted considering the advancements and validation achieved with SNALP technology.  Also, if Alnylam considered the subcutaneous approach to be that much more attractive than intravenous, you would think DPCs are the logical fall-back, if not more desirable than simple GalNAcs.  But maybe GalNAc Plus and DPCs are not all that dissimilar and we are on the same page after all.


Note added in proof (March 27, 2013): the ALN-TTRsc clinicaltrials.gov entry has now appeared.  As expected, however, no insights were provided into the dosing range. Only interesting tidbit: the volume of the placebo injection will be matched to that of ALN-TTRsc.  Injection volumes will be critical.

Arrowhead and Alnylam Vying for Subcutaneous RNAi Delivery Success

The use of the intravenous route of administration for the currently leading systemic RNAi delivery technology, Tekmira’s SNALP technology, has been noted to be a drawback of the technology, especially for non-severe diseases and in therapeutic areas historically dominated by oral medicines (e.g. the cholesterol-lowering market).  As a result, the arrival of two delivery approaches that promise to allow for subcutaneous administration has been welcomed: Arrowhead’s Dynamic Polyconjugates (DPCs) and Alnylam GalNAc-siRNA conjugates which have shown data suggesting their clinical use for gene knockdown in the liver (at least initially; DPC with potential to go beyond the liver).  

A day ahead of Alnylam’s Roundtable on conjugate delivery, I thought it would be a good time to get into the mood and compare the two competing technologies.

Basic Chemistries

GalNAc-siRNAs consist of siRNAs to which a cluster of three N-acetylgalactosamine residues have been appended.  It is these GalNAcs that are recognized by the ASGPR receptor protein that is abundantly presented on hepatocytes.  The choice of three over just one or two GalNAcs is due to the synergistic binding of multiple GalNAcs to the receptor.

DPCs also comprise of siRNA conjugates, but involve an additional endosomolytic agent to facilitate siRNA release from the endosomes.  The two components can be mixed together so that the drug can be given as a single formulation.  This, however, also requires that both siRNA and endosomolytic agent end up in the same place.  For hepatocytes, this is achieved by conjugating the siRNA to a cholesterol moiety and the endosomolytic agent to GalNAc.  

The reason why two different targeting agents are employed are two-fold: reduced competition for the uptake receptor, and not requiring triantennal GalNAcs such as in Alnylam's case which seems to involve a quite costly chemistry. The reason why GalNAcs on the endosomolytic agent in DPCs are not so expensive is because as a polymer (a peptide in the latest versions) multiple mono-GalNAcs can be conjugated distributively and still achieve the same synergistic binding effect.

Potency and Safety

The Holy Grail in RNAi subcutaneous delivery appears to be to get formulations potent enough so that the desired level of knockdown can be achieved with volumes of 1ml or less: you can squeeze only that much liquid under your skin through a thin needle.

The first of Alnylam’s GalNAcs, ALN-TTRsc, achieves a 80% target gene knockdown (ED80) following repeat administration in preclinical animal studies.  This is below the (based on OTS 2012) 3mg/kg barrier that apparently would allow for 1ml or less volumes in humans.  What surprised me to see at the OTS meeting in late October was that the GalNAc potencies, both in rodents and non-human primates, varied quite a bit between the programs.  The TTR formulation actually had the poorest potency among the programs.  This surprised me even more so given that ALN-TTR01 and ALN-TTR02 (both SNALP programs) contained highly potent RNAi triggers.  In the case of PCSK9, ED50 of less than 0.1mg/kg were obtained.

It is possible that the differences are not just due to the natural sequence-specific differences in RNAi potency, but a result of advances in chemistry.  In particular, optimizing siRNA-conjugates for tissue/endosomal stability rather than serum stability as is often practiced in RNAi Therapeutics is critical.  Importantly, this consideration also applies to DPC technology.      

DPCs should be more potent than isolated GalNAc siRNAs.  This is because you are adding an endosomal release agent to the liver-targeted siRNA (e.g. GalNAc-siRNA) conjugate and unfacilitated release of nucleic acid out of endosomes is believed to be highly inefficient.

Arrowhead has reported various impressive potencies such as 99% knockdowns at sub-1mg/kg siRNA doses.  This to me is strong evidence of the superior potency of DPCs over GalNAc-siRNAs.  Moreover, it seems that DPCs may inherently require less frequent dosing compared to GalNAc-siRNAs for which Alnylam aims at weekly or twice monthly dosing.

What is unclear, however, is the amount and resulting safety and volume implications of the endosomal release agent.  In particular, the most impressive knockdown data seemed to involve saturating amounts of endosomal release agent (~6mg/kg).  The first-generation endosomal release agent, PBAVE, suffered from relatively high toxicity, partly as a result of premature unmasking in the blood.  It makes sense that the newer, ‘more natural’ peptide-based endosomolytic release agents are safer.  By contrast, assuming that the GalNAc sugar itself is harmless, I am not too concerned about the safety of Alnylam’s GalNAc conjugates.

In terms of potency, advantage Arrowhead, in terms of safety, advantage Alnylam.

Strategic Considerations

The challenge for Arrowhead will be to make the case of the benefit of increased complexity over GalNAc-siRNAs. Would the prospect of a 3- or 5-fold increase in potency e.g. be enough justification for the investment?  I say ‘prospect’ because Alnylam could obtain knockdown proof-of-concept data at least a year before Arrowhead, especially since Arrowhead is planning to conduct the first study with DPC (ARC-520 for HepB) in healthy volunteers and thus won’t be able to measure viral target knockdown.

In addition to potency, DPC has the important advantage that it may be a more widely applicable RNAi delivery platform.  This alone may tempt others to put some money down on the technology to see where it can go.

Although GalNAc-siRNAs and DPCs are currently clearly competing, there is also scope for them to synergize, especially in the area of oligonucleotide chemistry.  Curiously, Alnylam did seek access to DPCs earlier this year, supposedly for its evaluation in one of its 5x15^TM programs.  Learning about DPC siRNA chemistry may be of at least equal, if not considerably more value to Alnylam.

Which of the two delivery technologies do you prefer for target gene knockdown in the liver?  Take the survey on the top right-hand corner.