Regado Shock Raises Questions for Oligonucleotide Therapeutics Field

Ave biotech investor, morituri te salutant.

Last week, aptamer company Regado Biosciences imploded in a spectacular fashion after it had to permanently terminate a large pivotal phase III trial of its lead therapeutic program, REG1.   It followed notification that an unacceptable rate of serious anaphylactic adverse events had occurred.

Aptamers are oligonucleotides that recognize their protein targets based on their 3-dimensional shape, instead of sequence complementarity as is the case with most other oligo-based mechanisms of action.  

Because REG1 involves oligonucleotides and pegylation chemistry, the event warrants the close attention of the wider oligonucleotide therapeutics field.  This blog tries to summarize what is known about REG1 and the adverse events and aims to pinpoint potential safety landmines as well as suggest strategies to circumvent them for the benefit of ongoing and future oligonucleotide drug candidates.

REG1 a 2-component system for the tight regulation of hemostasis

The bane of anticoagulant drug development is that too much of it and/or inhibiting clotting at the wrong drug targets can lead to great morbidity and death due to uncontrolled bleeding.  REG1 aims to address this catch 22 by providing an oligonucleotide-based aptamer that binds and therefore inhibits a key regulator of the clotting cascade, Factor IXa, but with the twist that the inhibition can in turn be turned off at will by administering an antisense oligonucleotide that has sequence complementary to that of the aptamer.  This disrupts the 3-dimensional shape of the aptamer thus abrogating its ability to bind Factor IXa.

The aptamer (pegnivacogin) itself consists of a heavily modified 31mer oligonucleotide with lots of 2’-O-methyl and 2’-O-fluoro residues (~10kDa molecular weight).  However, the bulk of the drug is made of a 40kDa polyethyleneglycol (PEG) moiety appended to the 5’ end of the oligonucleotide.  

The antisense oligonucleotide functioning as the antidote meanwhile is an unassuming 15mer 2’-O-methylated RNA, hardly something that would catch the eye of an oligonucleotide toxicologist.

The reason why the aptamer had been modified with PEG was to extend its circulation half-life.  What otherwise would likely be a half-life of a few minutes for a simple 2'-O-methyl/fluoro oligonucleotide, obviously of little use for the intended regulation of blood clotting, could thereby be extended to over 24 hours when administered intravenously (à REG1) or an even more impressive ~1 week when injected subcutaneously (àREG2).

Serious allergic events in REGULATE PCI study

The REGULATE-PCI study that has just been terminated aimed to enroll approximately 13000 (!) subjects undergoing percutaneous (through the skin) coronary interventions (PCI) to unclog arteries around the heart.  Because things tend to clot around devices introduced into the human body and in the presence of physical stresses such as clot disruptions, such procedures have to be performed with the concurrent use of anti-clotting agents (anticoagulants).

The study was terminated when after about one quarter of the target enrolment (~3200), an imbalance in what the company described as ‘serious allergic adverse events’ in its press release was noted by the body tasked with ensuring the safety of clinical trial participants (DSMB).

Unfortunately, I was not able to retrieve a replay of the conference call held by the company to discuss the trial termination to gather more information on the nature of the adverse events.

It is, however, logical to assume that the serious allergic events are the same that had already been noted in the phase II RADAR trial.  Specifically, 3 of the ~500 subjects that received REG1 had allergic reactions ‘shortly’ after infusion of the pegnivacogin aptamer, 2 of which were considered ‘serious’ and in fact led to a stopping of the phase II trial after it was deemed that the study already had enough statistical power.  The affected patients were successfully managed with antihistamines and steroids (Povsic et al. 2013).

Immune reactions not foreign to Oligonucleotide Therapeutics

Immune stimulation, of course, is also the main safety risk of Oligonucleotide Therapeutics as a class.  We are all familiar with injection site reactions, alternative complement activations and attendant cytokine elevations, antibody generation to phosphorothioated oligonucleotides etc.  Some of them, e.g. TKM-Ebola most recently, have led to Clinical Holds or the discontinuation of drug candidates.  On the other hand, thousands of subjects have been given oligonucleotide therapeutics, some for years, with apparently acceptable safety profiles.

So is the oligonucleotide component of pegnivacogin the culprit for the ‘serious’ allergic reactions?

After reviewing the data, the safety issue is unlikely due to the aptamer itself.  A naked, in this case 2’-O-methylated, 2’-fluoro oligonucleotide alone is expected to be very safe, at least in terms of acute toxicity.  This conclusion can be drawn for example from the administration of naked, non-phosphorothioated oligonucleotides, including the systemic programs of Quark Pharmaceuticals. 

On the other hand, the reason for the apparent safety of unformulated oligonucleotides could be simply because such an oligonucleotide is rapidly excreted into urine and therefore has little chance to be toxic.  This is, however, unlikely since the allergic reactions were reported to occur shortly after drug administration, at a time when there would still be considerable concentrations of naked oligonucleotides left in circulation, therefore allowing for such a comparison.

It is interesting that only REG1 has been terminated so far, but not REG2.  Although this shoe could yet drop due to an abundance of caution, it suggests that the serious allergic events are dependent on the intravenous route of administration, a route of administration that is more generally plagued by immune-related ‘infusion reactions’ from small molecules to large biologics.

...enter Omontys...

This brings me to the topic of Omontys, aka peginesatide.  Omontys, of course, is a pegylated peptide that had to be withdrawn from the market 18 months ago following similar (rare) allergic events, including fatalities due to anaphylactic shock.  

Like pegnivacogin, the pegylation in Omontys had a molecular weight of 40kDa.

Like pegnivacogin, there were both intravenous and subcutaneous versions of peginesatide, but only the intravenous version was associated with the severe allergies that occurred shortly after drug administration.  

Like pegnivacogin, the allergies only occurred during the first administration of peginesatide (note: for pegnivacogin there is just 1 administration).

So taken together with the well-known hypersensitivity often seen in response to PEG, the evidence strongly points towards PEG as the culprit, not the oligonucleotide.

Pegylation widely used in RNAi Therapeutics

This unfortunately does not entirely exonerate oligonucleotide therapeutics.  The reason is that pegylation is a widely used tool in the biotech industry, and within Oligonucleotide Therapeutics, RNAi Therapeutics has made ample use of it and promises to support advances in delivery, especially beyond the liver. 

This raises the question of whether such problematic compounds can be spotted earlier.  If not, or if the decision is to continue development, can simple allergy tests identify the subjects that should not get the drug, or would anti-histamine and/or steroid pre-treatment be practical and acceptable?  Or is it even just a manufacturing/quality control issue or a matter of the molecular weight of PEG? I’m sure some of these questions sound familiar to the readers of this blog, and I expect that the field will learn over time if and when to use these and other strategies. 

As both fearlessness and fear can be fatal to drug development and investment returns, it is worth reminding ourselves here that numerous pegylated drugs have been approved and are being commercialized.  Also, there are various degrees of allergic events, some more serious than others.  

Finally, as a former Affymax (and Lehman Brothers in case you wanted to know) investor who lost a few feathers, I and Affymax just were incredibly unlucky.  It is my contention that Omontys would be widely used today if it had not been for the conservative market entry strategy by Omontys-distributor Fresenius Medical Care which involved the close monitoring of the first commercial Omontys patients which picked up the very rare events.  

Also, why simple strategies such as the use of anti-histamines or just going with the subcutaneous version were not attempted to salvage Omontys remains a mystery to me.  Blame it on the damaged goods theory.

  

For the sake of Regado investors and medicine, let’s hope that the parallels end here and Regado won’t follow in the footsteps of Affymax to bankruptcy and class action lawyers.  

Implications of Ebola data for Tekmira HBV Program

Like it or not, it is the HBV program that is driving the interest in Tekmira, at least in the near-term.  Therefore, the 15% sell-off in the stock yesterday following the Clinical Hold imposed by the FDA can be interpreted to be due to the uncertainty of what it means for the HBV program.

It is my working hypothesis that for the treatment of HBV via the immune reactivation pathway, the use of even transient immune suppression is to be avoided.  On the other hand, the recent Ebola clinical results by Tekmira suggest that transient immune suppression may also be desirable for 3^rd gen SNALP delivery.  As TKM-HBV is expected to utilize '3^rd gen' SNALP delivery, too, the Ebola results with immune activation seem like a bad omen.

While this cannot be ignored, there are a few reasons to believe that this might be too simplistic.

1.       Difference in dose

Although there was an apparent tendency for immune stimulation throughout the dose escalation with TKM-EBOLA, it was only at the high dose of 0.5mg/kg where a dose-limiting toxicity was observed. And according the yesterday's press release on the Clinical Hold, it is in particular cytokine elevations that were observed at 'higher doses' that are the issue.  

 Since unlike Ebola, HBV only replicates in hepatocytes, and because SNALP potency is highest in hepatocytes, the efficacious dose for HBV may be well below 0.5mg/kg with 3^rd gen formulations.  Of note, even 2nd gen SNALPs are highly effective at 0.3mg/kg based on the Tekmira-enabled ALN-TTR02 candidate now in phase III.

2.       Different sequence, different immune stimulation

It is known (e.g. Judge et al. 2005: Sequence-dependent immune stimulation of the mammalian innate immune response by synthetic siRNAs) that immune stimulation by RNAi, especially when liposomally formulated, is highly dependent on the actual sequence and modification pattern of the RNAi trigger.  Since TKM-EBOLA and TKM-HBV are different in that regard, it is well possible that while one 3^rd gen formulation is immunostimulatory, the other is not.

3.       Immunostimulatory by design/by screen

Scientifically, I was most disappointed by the Ebola immune stimulations as Tekmira made a big deal in the wake of similar observation in 2010 with TKM-ApoB that with improved, more predictive immune stimulation assays, they would now be able to screen out the immunostimulatory formulations.  In a black-and-white world you would conclude ‘obviously not!’.

In a world of shades of grey, I can imagine that TKM-EBOLA might have happened to slip through this part of the screening process.  The reason is that since Ebola infects a number of cell types besides hepatocytes (endothelial cells, phagocytes etc), the impressive efficacy of TKM-EBOLA always stood out as something quite unusual.  So while containing the spread of the virus by hitting it particularly hard in the liver might be part of the mechanism, I am quite receptive to the notion that the scientists did not mind all that much some immune stimulation when the RNAi formulation had so much efficacy.

With HBV, however, the company might be much more careful in screening out immunostimulatory potential.

I can now hear Sarepta investors yell ‘it’s an artefact’, but I would caution them that Sarepta and the morpholino field at large is proposing morpholinos with cationic appendices for infectious diseases while these are not really considered for genetic diseases.  What I don’t understand is that why a modification that is supposed to be helpful in e.g. suppressing viral gene expression should not likewise be useful for suppressing human gene expression.  So maybe the value of these modifications is in tickling the immune system?

4.       Not all 3^rd gen are created equal

In response to a questioner in the Q1 financial conference call, the CEO of Tekmira said that the definitions of the various SNALP generations is not as clean as you would like from a chemical point-of-view.  Instead, the definition largely is a reflection of the potency improvements of the formulations.  Therefore, since TKM-EBOLA has a different set of target cells than TKM-HBV, it is well possible, even expected, that the formulations will also differ in their lipid compositions.  Since lipids are an amplifier of the liposomal-RNAi immune response, differences therein could have big implications as to the immune stimulation of the various formulations.

Disclosure: I am long Tekmira and only partially heeded my own advice of stepping out of the TKM-Ebola ‘Haertetest’ (trial provided not much upside to investors).  On the other hand, Tekmira has upside potential from a number of non-Ebola programs especially HBV and mRNA delivery.  As the worst case scenario, an outright termination of TKM-EBOLA, is a real risk, it becomes more and more difficult to get the timing right ahead of the upcoming TKM-HBV data.  If, however, you don’t mind volatility and are a long-term investor, it’s another matter.  However, at least for me, Tekmira is not the stock any more to maintain a 100% position in.

Alnylam Says Adverse Event in Tekmira ApoB Trial Was ‘Life-Threatening’

One apparent tactic employed by Alnylam in the ongoing litigation with Tekmira is not to win on arguments, but by coming out with 'revelations' aimed at unsettling Tekmira investors and potential collaborators. A weakened share price and financial position would supposedly make Tekmira more amenable to a light settlement or lowball takeover offer. Much of this has been discussed on this blog before, but today I would like to bring to the attention of the readers here one particularly grave and damaging remark that Alnylam made in their Response to the Amended Complaint, namely that the one case of immunostimulation in the phase I ApoB study that caused Tekmira to stop the trial was ‘life-threatening’:

From the Response:

‘Further, Alnylam invited Tekmira to join a joint development committee that Alnylam had formed with one of its pharmaceutical partners with the goal of assisting Tekmira and the whole field in advancing LNP technology. Moreover, it is through clinical trials conducted, mainly by Alnylam, that critical elements of Tekmira’s siRNA delivery technology have been validated in the clinic, and Alnylam has provided critical advice and counsel to Tekmira related to their pre-clinical and clinical development activities for their own products. For example, Alnylam’s chief medical officer provided critical and urgent counsel to clinicians attending to a patient in a Tekmira clinical trial that experienced a serious, life-threatening adverse reaction to Tekmira’s drug [emphasis mine].'

Not only does Alnylam make it sound like they are holding little Tekmira’s hands, have the new Chief Medical Officer rush to inept Tekmira's help, and more or less run the trials for them, this statement essentially accuses Tekmira of lying to investors and in front of regulators about the adverse event. These are very serious allegations, some of which by the way were part of the rumor mill even before Alnylam made these statements now in public (!).

These characterizations are in sharp contrast to the way Tekmira used to describe them: serious enough to be taken into consideration for future SNALP development, but not a show-stopper and certainly not life-threatening. Here is how:

Disclosure of adverse event in the phase I ApoB press release:

‘The primary endpoints of the ApoB SNALP Phase 1 clinical trial were measures of safety and tolerability. ApoB SNALP was well tolerated overall in this study with no evidence of liver toxicity, which was the anticipated dose-limiting toxicity observed in preclinical studies. Of the two subjects treated at the highest dose level, one subject experienced flu-like symptoms consistent with stimulation of the immune system caused by the ApoB siRNA payload. The other subject treated at the highest dose level experienced no side effects. Based on the potential for the immune stimulation to interfere with further dose escalation, Tekmira decided to conclude the trial [emphasis mine].’

If the adverse event had been ‘life-threatening’, then not disclosing this in the press release and subsequent investor discussions would obviously have been misleading.

The clinical details of the adverse event were then disclosed at the Drug Information Association '3rd Oligonucleotide-based Therapeutics Conference' held in Bethesda on March 23, 2010. An important function of these DIA meetings is to further the dialogue between industry and the US regulatory authorities and I assume that the FDA was in attendance and that Tekmira assumed the same when it reported the following:

‘Adverse Event in Suject 190:

- 53 Year old 103 kg male w/ hyperlipidemia

- Second in cohort to be treated with 0.6 mg/kg

- Tolerated infusion well

- ~2.5 hours felt ‘wobbly in legs’

- ~4 hours rigors, vomiting, fever, hypotension, hypoxia, HR 110 BPM

- Treated with Ibuprofin, O2, saline infusion

- Fever resolved in 3 hours

- ~5 hours BP reached nadir

- Patient continued to receive fluids

- IV methylprednisolone (120mg)

- BP, HR, O2 Sat improved

- Patient normal that night

- Returned to study unit the next morning

- Discharged the next day as per protocol

- Patient maintained perfect cogntion throughout episode

Event described as moderate in severity and related to study drug [emphasis mine].'

The account above is consistent with characterizing the event as ‘moderate’ and in no way suggests a ‘life-threatening’ event. While not exactly encouraging, such safety findings are common in clinical trials.

I’m confident that if this comes to trial, this damaging characterization of the ApoB trial will add another few millions to the damages awarded to Tekmira. I simply cannot believe that Tekmira’s management would risk getting in trouble with the SEC and FDA in such a way. There simply is no middle-ground. I know Tekmira shareholders are getting tired of hearing this, an attitude attesting to the success of Alnylam's tactics.

In related News: Alnylam guides down cash as ALN-VSP02 wraps up

Alnylam reported Q2 financial results after the close yesterday. Months after the departure of the former CFO and following the $150M shelf filing, the replacement had the pleasure of announcing that Alnylam is guiding down cash from ‘greater than $275M’ guided 3 and 6 months ago to now ‘greater than $250M’. No explanations were provided for the implied 50% increase in net burn. I hope that the new CFO has retained his ‘sense of humor’- apparently a job requirement at Alnylam [correction 5 August, 2011: in the conference call that followed the press release of the financial results the increase in expenditures were attributed largely to the 5x15TM efforts; not very illuminating really, but there was an explanation].

On the day of the financial results, Alnylam also disclosed that it has completed the phase I study in liver cancer with ALN-VSP02. There was no real new development in this study from the comprehensive data presented at this year’s ASCO (related blog here). What I noticed though is that this drug candidate has now been fully renamed ALN-VSP, dropping the '02' which may have been a painful reminder that Alnylam got all its clinically-relevant LNP delivery from Tekmira [hint: my strong impression based on the VSP conflicts that emerged 4 years ago with the remarks by David Bumcrot is that ‘01’ was a lipidoid formulation, and the ‘02’ formulation resulted from Alnylam giving up and seeking help from Protiva (now part of Tekmira, of course)].

Developing Multi-Functional RNAi Therapeutics

The great attraction of developing therapeutics based on RNAi is its inherent specificity and applicability to virtually any gene for the rational design of drugs. The specificity of gene silencing is not just a theory, but very powerfully attested by the many successes of applying RNAi even to genome-wide screens to uncover gene function (and also more targets for RNAi). Delivery of the RNAi inducer to the cells of interest would further add to such specificity and consequently safety.

However, as has long been known, certain nucleic acids elicit immune responses, and siRNAs are no exception to this. Although this does not apply to appropriately designed and vetted siRNAs, when it does, it may well interfere with the interpretation and predictability of the knockdown phenotype. However, instead of describing once again methods whereby such responses can be avoided and phenotypes more consistently obtained (siRNA length, structure, and modifications; bioinformatics etc), I would like to take the opportunity here to point out the potential for RNAi Therapeutics that include a immune-regulatory element. Moreover, as this is typically related to the uptake of the RNAi formulation in cells other than the primary target cells, I would also like to make us consider the potential for RNAi Therapeutics exploiting the entire biodistribution of a particular RNAi drug delivery system.

When we think about indications such as cancer and viral infections, the importance of the immune system in eliminating the disease cannot be underestimated. Cytokine therapies e.g. are well known to these areas of medicine and it is no coincidence that there have been long-standing efforts in harnessing the ability of nucleic acids to induce TLR and other immune signaling pathways to improve both cellular and humoral immune responses.

It is therefore conceivable that an immuno-stimulatory siRNA is not necessarily screened out during the siRNA selection process, but is deliberately packaged into a nanoparticle which in addition to the primary target cells (cancer cell, virus infected cell, etc.) would also be taken up by phagocytic cells where the cytokine stimulation would lead to enhanced antigen presentation or the augmentation of monoclonal antibody therapies. To further take full advantage of the biodistribution of the RNAi formulation, the RNAi drug could also contain two or more different siRNAs, each one designed to knock down a suitable gene in the various cell types that the nanoparticle is taken up in (e.g. in the case of a liver delivery system that enters both Kupffer cells and hepatocytes, an siRNA against a immuno-regulatory gene expressed in the Kupffer cells and maybe other phagocytes and one siRNA for a hepatocyte-specific gene).

In addition to immune-stimulation, certain siRNA formulations could be used for concomitant gene knockdown and immune suppression. As work by Protiva (now Tekmira) has shown, siRNA modifications may not only be used to avoid unwanted TLR signaling through siRNAs, but to inhibit these TLR responses in trans. A single modified siRNA could thus be employed in a two-pronged gene knockdown/TLR signaling inhibition strategy for treating autoimmune disorders.

Based on the acquisition of prior TLR company Coley by Pfizer, Alnylam’s vaccine spin-off intentions, and Tekmira’s IP and know-how on the immunological properties of nucleic acids, I would not be surprised if we should be hearing relatively soon more about such multi-functional RNAi Therapeutics.

Rather than considering innate immune responses and imperfect biodistribution as nuisances, it may well turn out that a number of RNAi Therapeutics may get the extra bit of efficacy out of simultaneously modulating immune responses and knocking down genes in multiple cell types. In my opinion, the medical and commercial opportunities for that are currently underappreciated.