Tekmira and Alnylam in Binding Arbitration Over $5M ALN-VSP Milestone Payment

The Alnylam-Tekmira dispute may not be over yet.  After de facto losing in the trade secret litigation over SNALP delivery technology, $3B heavy weight Alnylam seems intent on making life for Tekmira difficult and earn the milestones it is entitled to according to the settlement the hard way.

At least this is how I interpret the disclosure in the 10-Q quarterly report filed today with the SEC by Alnylam where the company discloses that it has entered into binding arbitration with Tekmira over a $5M milestone payment related to liver cancer candidate ALN-VSP02:

‘In addition, we recently initiated binding arbitration proceedings to resolve a disagreement with Tekmira regarding the achievement by Tekmira of a $5.0 million milestone payment under our cross-license agreement relating to the manufacture of ALN-VSP clinical trial material for use in China.’

As a reminder, Tekmira was to earn the milestone for enabling Alnylam’s ALN-VSP partner Ascletis to enter clinical development in China. 

I do not wish to speculate on the specifics of the dispute, but it is worth remembering that Alnylam is heavily dependent on rapid clinical development of SNALP-enabled ALN-TTR02 to support its valuation, especially since the GalNAc platform is showing additional blemishes (ALN-AT3 delay; admission in the conference call of certain side effects seen with ALN-TTRsc).

RNAi Therapeutics Arriving in Oncology

Yesterday, Tekmira presented data from the dose escalation portion of the phase I trial with TKM-PLK1 for the treatment of solid tumors.  I agree with the company that the fact the drug candidate was associated with clinical benefit in 4 out of 9 evaluable patients receiving 0.6mg/kg or more versus no such association at dosages below is 'highly suggestive' that TKM-PLK1 has anti-tumor activity indeed.  I also agree with their decision to pursue the neuroendocrine tumor indication for their first phase II study later this year based on the present data.

What I would like to focus on today, however, is much more exciting data contained in the TKM-PLK1 data as it relates to RNAi delivery to oncology…the key obstacle in making RNAi Therapeutics work for cancer.  Specifically, the long-circulating 2^nd gen SNALP formulation employed in TKM-PLK1 exhibited vastly improved pharmacokinetics over the short-circulating 1^st gen SNALP formulation employed in Alnylam’s ALN-VSP02.  Whether cancer in the liver or not, long circulation times are critical in order to take advantage of the Enhanced Permeability and Retention (EPR) effect that is at the heart of nanoparticle-based approaches in oncology.

It is for this fact that I am optimistic that the ‘highly suggestive’ data (which you invariably will hear about in phase I cancer studies involving a mix of late-stage cancer patients such as this one) are for real.

10-Fold More siRNA at 24 hours

Slide 13 of the AACR presentation shows that at 24 hours, around 1500 nanograms siRNA was present in 1ml of serum at the maximally tolerated dose of 0.75mg/kg.  This compares to around 150 nanograms of combined KSP and VEGF siRNA per ml at this time point for ALN-VSP02 as presented at ASCO2011.  If you’ve ever been at the bench and performed siRNA transfections in tissue culture cells, 1.5 microgram siRNA in a ml of media is overkill, and in this case it is real tumors that are served by blood containing such large siRNA concentrations- for days.

Considering the PK profile, it is not surprising that the one paired tumor biopsy obtained so far showed RNAi activity according to 5’ RACE, whereas such detection was rare in the ALN-VSP02 trial.  Clearly, more tumor biopsies have to be taken in the dose extension phase of the study (which apparently is recruiting quite well).  This will also be important for assessing siRNA concentrations in the tumors themselves.

Selecting the Right Patients

A heterogeneous patient population as in this trial alone obviously makes it very difficult to select the right cancer types for future clinical development.  There are a number of biomarker strategies that Tekmira could, and in my opinion, should employ in their future development.

Based on target biology, the KRAS mutation status should be given high priority.  KRAS-driven cancers are thought to be highly dependent on PLK1 expression.  Related to this, PLK1 expression itself could be used as a guide in patient selection.

A more unusual strategy would be to use imaging technologies based on nanoparticles which presumably rely on the same EPR effect in order to assess whether the tumors are amenable to EPR effect (alternatively, a simpler perfusion test may provide similar insights).  I could imagine that the fact that neuroendocrine tumors seem to respond to TKM-PLK1 is a result of such a favored effect.  Finally, another (admittedly less likely) selection strategy considering delivery would be to target tumors with high expression levels of LDLR-like receptors which are thought to be important in the cellular uptake of SNALP particles.

And last but not least, for those that like to think of comparative values, with a much better target and PK profile over ALN-VSP02, TKM-PLK1 may now be considered the lead RNAi Therapeutics oncology candidate. 

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Cancer RNAi Therapeutics Moving On

As RNAi Therapeutics targeting genes expressed in the liver, especially for orphan indications, have captured much of the recent interest in the technology, it is time to once again pay closer attention to RNAi Therapeutics in the oncology space.

Atu027 and TKM-PLK1 Moving into Phase II

Silence Therapeutics have just announced that they got the green light from the German regulators to test Atu027 in combination with small molecule gemcitabine in pancreatic cancer.  Following a short phase Ib to establish the safety of the combination (note: the original phase I was a monotherapy trial), the plan is to rapidly move into phase II which will aim at establishing proof of concept for efficacy.  Depending on the funding situation, it is possible that the company will expand Atu027 into other indications in phase II, possibly in collaboration with private investigators in the UK.

Clearly, the strength of Atu027 is its safety profile so far, one that has surpassed my own expectation for this positively charged lipoplex formulation.  The clinical attraction meanwhile is that Atu027 aims at a quite novel anti-cancer mechanistic approach by inhibiting metastatic spread by fortifying the vascular endothelia. 

The challenge with Atu027, however, is that the gene target, PKN3, remains a relative black box.  This not only demands a certain leap of faith in mechanistic terms, but also complicates the clinical development as biomarkers and other tools are not readily available.

This is quite different from TKM-PLK1, the cancer RNAi Therapeutics by Tekmira.  Polo-like kinase 1 is a hot target, but specificity/safety issues have so far dogged drug development efforts with small molecules, most notably represented by Boehringer-Ingelheim.  By contrast, the SNALP RNAi delivery approach promises to be more cancer tissue targeted and consequently should avoid some of the dose-limiting toxicity observed so far, especially the hematologic ones.

TKM-PLK1 has flown a bit under the radar and the phase I update late last year did not capture much attention.  This is probably because the response rate did not seem too much to get excited about.  However, as Ian MacLachlan, the CSO of Tekmira, pointed out at the AsiaTides meeting last week, it was a small dose-escalating phase I trial (20-30 patients) in advanced solid cancer patients and only four patients actually received TKM-PLK1 for two or more cycles (6 infusions over 2 months) at 0.6 mg/kg or above, dose and durations you would think would be required for advanced cancer patients to get a chance to respond in the first place. Notably, two out of those four showed signs of efficacy: one partial response and one with stable disease after more than 6 months on study drug.

In light of that, the phase I profile of TKM-PLK1 supports my view that it could be the foundation for an exciting PLK1-related cancer franchise for Tekmira and I look forward to seeing the full results presented at the upcoming AACR meeting.  My attention will be particularly focused at any direct measures of target knockdown.  Beyond that, with the company’s healthy financial situation, I look forward to a rich phase II strategy involving a number of solid cancers and innovative study designs such as patient selection based on genetic criteria.  As good as Tekmira's science is, they now also need to be as proactive and innovative on the clinical front.

ALN-VSP02 and CALAA01 Falling Behind

As TKM-PLK1 and Atu027 are moving into phase II, fellow early movers in oncology RNAi ALN-VSP02 and CALAA01 are falling behind. 

The next step for ALN-VSP02 will be a phase I in hepatocellular carcinoma (‘real’ liver cancer) in China under a license by Alnylam to Ascletis.  As noted by Patrick Lu of Sirnaomics at AsiaTides though, it would be a mistake to believe that the Chinese regulatory environment is any less demanding than in the West.  Au contraire, it seems that much more paperwork is required to file an RNAi-realted IND as the authorities there are keen on building their expertise in this new technology.  Expect a delay of at least 6 months from IND application to the green light to go ahead, after all the paperwork has been submitted.  For Tekmira watchers, I expect the $5M milestone thus in 2014, not in 2013.

While ALN-VSP02 still has a pulse, despite of the fact that it lacks the long circulation thought to be necessary for cancer uptake (note: delivery to liver cancer is a very different animal from delivery to normal liver; TKM-PLK1 is a long circulating SNALP), RIP CALAA-01 by Arrowhead Research.  Having said that, the value of CALAA-01 to Arrowhead Research has not been lost entirely as the company has filed for a patent covering a low/high dose strategy of minimizing immune stimulation.  

As the recent high-profile pulling from the market of a pegylated peptide (Omontys by Affymax) due to hypersensitivity, even anaphylactic reactions demonstrate (and also the infusion reactions reported by Alnylam in their SNALP RNAi Therapeutics trials), administration-related hypersensitivity reactions are an issue for the entire drug development arena.  In order to rescue the value of a number of blockbuster franchises, I expect that changing administration schedules could be one area attracting increased attention.  A patent here could have unexpected value.   

A New Crop of Cancer RNAi Therapeutics

Behind TKM-PLK1 and Atu027, a new crop of cancer RNAi Therapeutics is emerging.   Dicerna has signaled that it will finally move into clinical development (Q1 2014) with a liposomal Dicer-substrate formulation with a focus on liver cancer.  Of note, the liposomal formulation process is quite distinct from that of SNALP involving sequential core and envelope formation steps, potentially amenable to ligand-directed targeted delivery.

Another program worth following is another one for liver cancer, the subject of a collaboration of Korean company Bioneer andSanofi-Aventis.  The attraction of that program is that the nanoparticle here, SAMiRNA, comprises a single molecule, even to the extent that the RNAi trigger and delivery components can be manufactured on a single column.  Although I’m opposed to the fundamental notion that the ease of route of administration and the manufacturing process should drive drug development, don’t underestimate the attraction of meeting idealistic product profile when it comes to partnering with Big Pharma.    

The efforts above are a reminder that cancer could be an even bigger commercial opportunity for RNAi Therapeutics than liver-related indications.  The genome-wide targeting opportunity with RNAi Therapeutics has not been lost.  The pharmaceutical industry is watching.

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ALN-VSP02 Was a Good Start, but Directs Focus Towards Improved Formulations

Alnylam recently published detailed phase I results from its RNAi Therapeutic candidate to treat liver cancer, ALN-VSP02.   Although results from this study had already been reported, the new paper by Tabernero and colleagues provides an insightful warts-and-all view of Tekmira's SNALP generation 1.0 (short-circulating DLinDMA particles) in a solid cancer.  The results show that while ALN-VSP02 did achieve a number of important goals, including evidence of tumor response and RNAi activity, it will be necessary to turn to the newer SNALP formulations for more robust efficacy and improved safety.

New Insights on ALN-VSP02 Activity

Previously, Alnylam provided clear evidence of RNAi activity using the 5' RACE assay.  It is now clear, however, that this was only based on 3 positive biopsies out of 15 investigated in this regard, and only for the VEGF, but not the KSP target gene.  Consistent with this less than robust RNAi activity, there was no convincing evidence of target gene knockdown and no spindle abnormalities as would have been expected from KSP knockdown.

New Insights on ALN-VSP02 Safety  

The present paper also provides valuable insights into the safety of a SNALP 1.0.  As was reported before, infusion reactions were seen in a minority of patients and this was readily addressed in practical terms by slowing the rate of infusion.  Interestingly, the infusion reactions appear to be related to complement activation, not cytokine activation.

Some cytokines were also upregulated (rarely TNF-alpha, however), especially above 0.4mg/kg, despite the use of modified siRNAs.  The evidence thus increasingly points towards the DLinDMA lipid itself to be the culprit.

Finally, ALN-VSP02 caused considerable spleen toxicity, especially with prolonged dosing.  In the one complete responder in the study, 90% of the spleen was reduced after receiving 50 doses of ALN-VSP02.  This is consistent with the preclinical monkey data.  Somewhat reassuringly for SNALP 1.0, this effect might be due to on-target efficacy, as the same lipid formulation with a luciferase siRNA did not have this effect in monkeys (note: the spleen is not an essential organ in humans, and I’d much rather have my spleen removed than cancer).

SNALP-based Cancer RNAi Therapeutics in the Future

Based on the above limitations, it is important that future SNALP-based cancer RNAi Therapeutics include the following improvements, essentially all of which have been achieved over the last 6-7 years since ALN-VSP02 had been selected as a development candidate: firstly, a more potent and less immunogenic lipid is required (there should be multiple to choose from now); secondly, the SNALP particles should be less than the 80-100nm with ALN-VSP02 and include more stable PEG-lipids for improved, i.e. prolonged circulation times to better take advantage of the EPR effect of solid tumors (TKM-PLK1 includes such stable PEG-lipids); and finally, as spleen safety might also be affected by lipid accumulation along the endosomal-lysosomal pathway, enhanced lipid biodegradability may be desirable.

ALN-VSP02 has been put on hold by Alnylam for development in the Western markets.  Last year, however, Alnylam licensed development and commercialization rights to Ascletis for the Greater China market where liver cancer (hepatocellular carcinoma, HCC) is prevalent.  The next clinical study by Ascletis is expected to involve more HCC patients who, unfortunately, have been almost entirely missing in the phase I study of ALN-VSP02.  Despite the challenges that ALN-VSP02 will face in HCC, innovative RNAi Therapeutics have an important role to play in the future treatment of HCC, a disease that is proving again and again hard to manage by more conventional approaches as Celsion’s disappointing results (radiofrequency ablation with liposomal doxorubicin) today showed. 

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.

ASCO 2012: Silence and Alnylam Provide Update on RNAi Therapeutics Candidates for Solid Cancer

It is this time of the year again when abstracts for the upcoming ASCO mega-cancer meeting are released (ASCO 2012 in Chicago, June 1-5).  Among the poster abstracts were phase I updates for Atu027 by Silence Therapeutics and ALN-VSP02 by Alnylam for advanced solid cancers and cancers with liver involvement, respectively.  Although the full details are yet to be presented at the meeting and the abstract date was in early February (this particularly means that critical data from the high-dose cohorts for Atu027 are missing), I will post here the abstracts as appetizers, each one followed by some brief comments. Highlights are mine.

 

Abstract No. e13597 by Silence Therapeutics

Antimetastatic activity of Atu027, a liposomal small interfering RNA formulation, targeting protein kinase N3 (PKN3): Final results of a phase I study in patients with advanced solid tumors.

Sub-category:
New Targets, New Technologies

Category:
Developmental Therapeutics - Experimental Therapeutics

Author(s): Dirk Strumberg, Beate Schultheis, W Meyer-Sabellek, C. Vank, F Gebhardt, A. Santel, O. Keil, K. Giese, J. Kaufmann, Joachim Drevs; University of Bochum, Marienhospital Herne, Department of Internal Medicine III, Herne, Germany; Silence Therapeutics AG, Berlin, Germany; Tumorzentrum UniFontis am Eduarduskrankenhaus, Köln, Germany

Background: Atu027 contains siRNA-lipoplexes, which elicits RNAi mediated suppression on PKN3 in vascular endothelial cells. In various xenograft mouse models, silencing of PKN3 expression and significant inhibition of invasive growth, lymph node and pulmonary metastasis formation was shown. Methods: Atu027 was applied to patients (pts) as a single 4h-infusion with subsequent follow-up for 3 wks. Thereafter pts were treated twice weekly for 4 weeks. In case of SD, pts were treated until PD. Dose escalation was associated with assessment of toxicity, pharmacokinetics (PK), and multiplex biomarker analyses in plasma from treated pts. Results: A total of 33 pts have received Atu027 of 11 dose levels (DL) up to 0.336 mg/kg. No pre-medication was required. No cytokine activation (TNF-α, IL-1β, IFN-γ, IL-6) was observed. In some subjects transient activation of the complement system (C3a, Bb, sC5b-9) was found, but without any clinical relevance. PK-data showed dose-dependent increase in plasma siRNA as well as lipid levels. Among various biomarkers tested, sVEGFR-1 plasma levels decreased significantly upon treatment. Across all dose levels, Atu027 was well-tolerated. Adverse events possibly related to Atu027 were fatigue grade G1 (6pts), hair loss G1 (2pts), sweating G1 (1pt), and abdominal pain G2 (1pt). G3 AEs not considered as DLTs were elevated lipase (2 pts, DL2+DL10) and diarrhea (1 pt, DL5). So far, no DLTs were seen in the last DL. Stable disease after 3 and 6 months was observed in 10 and 3 pts, respectively. Two pts with neuroendocrine cancer had disease stabilization for 9 and 12 months, respectively, including partial regression of pulmonary metastases in 1 pt. Another patient with breast cancer had regression of liver metastases.Conclusions: Atu027 is well-tolerated and anti-metastatic activity has been observed. Soluble VEGFR-1 might serve as a biomarker. So far, 0,336 mg/kg is the recommended dose for further phase II trials.

Comments: Importantly, dose escalation in this dose-finding study could proceed to the second highest planned dose level (0.336mg/kg), which at least based on the previously presented PK data should be well above the dose necessary (~0.25mg/kg) for obtaining solid target gene knockdown in endothelial cells.  As 0.336mg/kg was dose cohort 10 (up to 11 planned) and 33 patients have been dosed, this means that one cohort probably included 6 instead of 3 the subjects. I am curious to learn the reason for this in June and particularly will be looking for more insight on the complement activations which have been described to be ‘without any clinical relevance’.  Also of interest is that the soluble version of the VEGF-receptor 1 was found to decrease significantly upon treatment (maybe as an adaptation to an anti-angiogenic mechanism of Atu027?) leading the company to speculate that this could serve as a biomarker in future trials.  On the efficacy front, still nothing really conclusive can be said and there were no additional ‘hints’ of efficacy in the higher dose cohorts as one might have hoped.   Overall, however, I look forward to the updated and fleshed-out data in 2 weeks.

For a discussion of the Atu027 results at last year’s ASCO meeting, click here.

Abstract No. 3062 by Alnylam Pharmaceuticals

Sub-category:
New Targets, New Technologies

Category:
Developmental Therapeutics - Experimental Therapeutics

Author(s): Maria Alsina, Josep Tabernero, Geoffrey Shapiro, Howard Burris, Jeffrey R. Infante, Glen J. Weiss, Andres Cervantes-Ruiperez, Mrinal M. Gounder, Luis Paz-Ares, Rick Falzone, Jamie Hill, Jeffrey Cehelsky, Akshay Vaishnaw, Jared Gollob, Patricia LoRusso; Molecular Therapeutics Research Unit, Vall d'Hebron University Hospital, Barcelona, Spain; Dana-Farber Cancer Institute, Boston, MA; Sarah Cannon Research Institute, Nashville, TN; Virginia G. Piper Cancer Center at Scottsdale Healthcare, Scottsdale, AZ; Department of Hematology and Medical Oncology, INCLIVA, University of Valencia, Valencia, Spain; Memorial Sloan-Kettering Cancer Center, New York, NY; University Hospital - Virgen del Rocio, Seville, Spain; Alnylam Pharmaceuticals, Cambridge, MA; Karmanos Cancer Institute, Detroit, MI

Background: ALN-VSP02 is an RNA interference (RNAi) therapeutic comprised of lipid nanoparticle-formulated small interfering RNAs targeting vascular endothelial growth factor (VEGF)-A and kinesin spindle protein (KSP). In a phase 1 trial, ALN-VSP02 administered as an iv infusion q2 wks was well-tolerated and showed evidence of anti-VEGF pharmacology and antitumor activity. Methods: Patients treated on the phase I trial with stable disease (SD) or better after 4 months (8 doses) were eligible to continue on an extension study until disease progression. Main objectives included continued evaluation of safety/tolerability and assessment of disease response. Results: Seven of 37 patients (18.9%) evaluable for response went onto the extension study, including 1 of 7 (14.2%) at 0.4 mg/kg, 2 of 5 (40%) at 0.7 mg/kg, and 4 of 11 (36.3%) at 1.0 mg/kg. All had progressed after one or more prior therapies. Tumor types included head and neck squamous cell carcinoma, angiosarcoma, endometrial cancer, renal cell carcinoma (RCC, N=2), and pancreatic neuroendocrine tumor (PNET, N=2). At the time of enrollment, 6 had SD and one (endometrial cancer with multiple liver metastases) had an unconfirmed partial response (PR). The average length of time on treatment (including phase I and extension studies) was 9.5 months (range 5-19). As of January 2012, 3 patients remain on study, including the endometrial cancer patient with an ongoing PR who has had >80% tumor regression after 19 months of treatment at 0.7 mg/kg and two patients with RCC and PNET with continued SD after nearly 1 year of treatment at 1.0 mg/kg. The other patients with RCC and PNET at 1.0 mg/kg with SD came off after 8.5 and 5.5 months, respectively, for adverse events that included fatigue or elevated alkaline phosphatase. A decrease in spleen volume, likely an on-target effect and not associated with any adverse events, occurred to a greater degree on the extension study than on the phase I trial and was most pronounced in patients receiving ≥ 12 doses. Conclusions: ALN-VSP02 has preliminary activity against endometrial cancer, RCC and PNET and a favorable safety profile that permits chronic dosing. Phase II trials are warranted in these and other VEGF-overexpressing tumors.

Comments: Final results for this study were presented at last year’s ASCO meeting (for a discussion click here). The new data relate to the open-label extension phase which is of particular interest as it relates to the longer-term safety and tolerability of ALN-VSP02, and possibly first-generation SNALP delivery technology in general.  It is too early to tell to what extent siRNA sequence and modification-dependent effects played a role, but the apparent spleen toxicity is consistent with what one might expect from such formulations. Although the spleen is not an essential organ and most of us will do just fine without one, this could become a dicey issue for a chronically dosed drug like ALN-PCS02 in a ‘lesser’ disease such as many types of hypercholesterolemia.  Overall, however, this is an encouraging start for the longer-term safety of SNALP delivery technology.  Based on the indications of efficacy, another more focused study may be warranted before a final determination as to the viability of ALN-VSP02 for the treatment of cancers with liver involvement.

Alnylam Launches Pair of Pre-emptive PR Strikes to Cover Patent Losses

When the therapeutic relevance of a patent (Tuschl-I) that once had the potential to be broadly applicable to RNAi Therapeutics has been lost forever in Europe, and when in another US patent proceeding an important claim covering a cancer drug candidate that has just completed a phase I study has been declared invalid due to lack of written description support and may actually belong to your worst nightmare, it seems hardly a time to declare victories. Nevertheless, the PR department at Alnylam has risen to the challenge and done just that: two press releases (here and here) by the company shortly following these patent outcomes announced with great fanfare that Alnylam’s IP position had been bolstered. The reward for the skilled use of small qualifiers such as ‘requested’ and ‘key’ and obedience? Not just keeping your job, but actually being promoted to Vice President of Investor Relations.

Alnylam vs Silence Therapeutics et al.: Tuschl I in Europe

As Silence Therapeutics can attest, the Tuschl I patent estate, licensed by Alnylam and Merck, was once an effective deterrent against competing RNAi triggers of 21-23 nucleotides and/or base-pairs. While certainly an important contribution to the eventual discovery of RNAi triggers for use in human cells, its main vulnerability in IP terms derived from the fact that its research was mainly conducted in Drosophila fly lysates and tissue culture cells.

Consequently, in both the US and Europe its importance for RNAi Therapeutics has been waning to the point of being essentially relegated to the use of RNAi triggers isolated from Dicer processing reactions in the test tube (my bet is that we will never see such a product candidate). Nevertheless, some passages in the written description and claim language in the European T-I patent EP 1309726 was sufficiently ambiguous that it must have worried companies like Silence Therapeutics, BASF, and Sanofi-Aventis that it might be exploited by a legally aggressive company such as Alnylam as a convenient casus belli, causing them to oppose the patent. Specifically (former) independent claim 10 may have raised such concerns:

‘10. A method of producing knockdown cells, comprising introducing into cells in which a gene is to be knocked down isolated double-stranded RNA of from 21 to 23 nucleotides in length and corresponding to a sequence of the gene, that targets the mRNA corresponding to the gene and maintaining the resulting cells under conditions under which RNAi occurs, resulting in degradation of the mRNA of the gene, thereby producing knockdown cells.’

On February 29 and March 1 there was an unusually lengthy, and therefore probably highly contested Oral Hearing on the case, the outcome of which was quickly claimed by Alnylam (and patent co-owner UMass) as a victory. Particularly, it claimed that the ‘requested claims of the ‘726 patent were upheld without modification’ [emphasis mine]. Turns out that a comment poster on this blog last week got it right in that he/she pointed to the qualifier ‘requested’. To back up, essentially what happens in such an Oral Proceeding is that the Defending Party (here: Alnylam/Umass) submits alternative claim sets in case that the original claim language is rejected by the EPO based on the principle that a weaker patent is better than no patent at all (e.g. for the purposes of appearance).

After reading the now published documents from the Oral Hearing (if you have problems accessing them, you can request them by email from me), what happened is that Alnylam has indeed had the boldness to represent that after having been forced to modify the claims, the new claims were upheld in unmodified form. If you are an Alnylam investor/supporter, you may want to ask the new VP whether you are justified in feeling cheated by such an insincere conclusion and may want to re-think the general credibility of that company.

The upshot in terms of the patent: by clarifying the written description with expressions such as ‘isolating from combination’ and ‘in vitro’ and completely re-writing the claims, T-I is confirmed to be without relevance for RNAi Therapeutics. It remains important, however, for the commercialization of in vitro RNAi-based target discovery/validation services such as offered by companies like Cenix, although only for dsRNAs of 21-23nucleotides in length.

Alnylam vs Tekmira: Alnylam about to lose ‘key’ ALN-VSP patent

Similar to the T-I patent confrontation, Alnylam was quick to claim victory in yet another of its patent fights: ‘Alnylam retains key claims for Kinesin Spindle Protein (KSP) RNAi Patent in Interference Proceedings’. Briefly, last year the USPTO had called an Interference between an issued patent by Alnylam (US 7718629) covering an siRNA sequence in ALN-VSP02 (phase I studies completed) and a patent application by Tekmira which may in fact cover the same sequence.

As the UTPTO cannot grant identical claims in two different patents, the Interference proceeding will decide on the validity and priority of these claims. As Tekmira pointed out, this Interference has no relevance to that company’s pipeline. It could, however, mean that a patent covering a ‘key’ feature of ALN-VSP02 (an siRNA sequence) may not only turn out to be invalid, but actually belong to Alnylam’s worst nightmare: Tekmira.

By now, we all know the drill: look for the little qualifiers. ‘Key claims’ would be the obvious candidate here to home in on. But in this case, not even twisted logic can explain Alnylam’s conclusion, as in fact no claim (at least those contested and related to synthetic RNAi triggers) was ‘upheld’. Quite the opposite, the Appeals Board (BPAI) granted Protiva’s/Tekmira’s Motion 2 which requested that Alnylam’s KSP sequence claim be ruled unpatentable. The explanation was that while the claim covered dsRNAs between 15 and 30 nucleotides, only 19bp siRNAs with 2 nucleotide overhangs were presented in the application (--> lack of written description support). Since all the other claims relating to synthetic RNAi triggers are dependent on claim 1, I’m puzzled as to how Alnylam could ever claim that ‘key claims’ had been upheld, when in fact it looks like all the relevant claims will be revoked.

Claim 1 of US patent 7718629:

‘1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5) gene in a cell, wherein said dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence complementary to SEQ ID NO:1311, wherein said first sequence is complementary to said second sequence and wherein said dsRNA is between 15 and 30 base pairs in length.’

Protiva’s Motion 1 that Alnylam’s provisional applications be declared invalid as priority documents was denied. The importance of this ruling is that it may affect the outcome of the second stage of the Intererence proceeding which will be about determining priority. Because the provisional applications by Tekmira and Alnylam were filed only two months apart, with Alnylam having the earlier date, chances are that Alnylam would enjoy priority IF Alnylam’s ‘key’ claims were held valid. However, with the above ruling and a recent rejection of claim 32 in a pending patent application by Alnylam (serial number 13/165568), this seems highly unlikely (note: I am unable to see the pending patent application, but claim 32 should be directed at the KSP sequence). By contrast, the Appeals Board decided that, unlike Alnylam’s patent, the KSP sequence claims are supported in the written description in Tekmira's competing patent.

The upshot: Tekmira prevailed on all Motions (filed by Alnylam and Protiva), except for on the validity of Alnylam’s provisional applications for priority purposes and the Appeals Board deferring decision on including claim 32 from the pending patent application which, based on the March 6 rejection does not seem to help Alnylam much anyway. This means that not only are the ‘key’ sequence claims about to be rejected, but Alnylam’s worst nightmare may actually end up owning it.

Don’t take my word for it, you can read the relevant documents yourself here (T-I) and here (Interference). If you are somewhat familiar with reading such documents, it will only take you 30-45min to read (and understand) them each. It seems that Alnylam is relying on the fact that most news organizations and analysts cannot be bothered to read the primary documents and conveniently adopt Alnylam’s version of events. If you do a Google search for news related to Alnylam, you will see that this happens more often than not. After all, Alnylam has more money to feed into the biotech machine: why antagonize a lucrative source of income?

RNAi Therapeutics Financial Viability Looking Up Following String of Clinical Results

By: Dirk Haussecker

Note: A PDF version of this article is available at myfirstnameDOTmylastnameATgmailDOTcom

Abstract

Shortly after the 2006-8 period of exuberance during which access to capital was easy, the RNAi Therapeutics industry found itself in a financially difficult position. At the roots of this change were the eventual recognition of some poor science, clinical setbacks, and the tension arising from the more gradual progress of science and impatient markets. Clinical validation of RNAi-mediated gene knockdown following systemic delivery was seen as the only way out of this situation. This review summarizes how such critical validation was provided by a series of recent clinical results from the ALN-VSP02, Atu027, ALN-TTR01, and ALN-PCS02 development programs. These results are expected to reinvigorate investments in the technology.

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Background

In the spring of 2011, the RNAi Therapeutics industry had just gone through two financially very stressful years. A few large pharmaceutical companies (‘Big Pharma’), among them Roche and Pfizer, made high-profile exits from the space [1], pure-play RNAi Therapeutics companies were crashing, and optimism gave way to a sense that RNAi in humans may take much longer to achieve than once thought due to the delivery challenge and the risk of causing immune stimulations [2]. This negative view of the technology and its financial prospects came sudden. After all, it was only in 2006-8 that the medical importance of RNAi had been recognized with the Nobel Prize in Physiology or Medicine [3], and Merck and Roche alone accounted for almost $2B of investments in the technology.

The argument can be made that the RNAi Depression was catalyzed by the US Housing Crisis and the ensuing global financial turmoil towards the end of 2008. This triggered a retrenchment of capital from high-risk innovation, capital that failed to return to the RNAi space. The seeds, however, had already been planted during the RNAi Therapeutics hype phase. As often is the case in such periods, it attracted the attention of the notorious promotional biotech schemes which in turn were readily followed by the fast money crowd, all of which, of course, did not mix well with the more gradual pace of technological progress. The scientifically leading pure-play RNAi Therapeutics companies can be accused as well for having catered to the cravings of fast money by over-promising on timelines and pushing programs into the clinic which were not adequately mechanistically validated (e.g. clinical candidates based on the local administration of naked, sometimes entirely unmodified siRNAs to the eye and respiratory epithelium). Similarly, the scientific community and journals can be blamed for failing to better police the quality of the science that got published [4,5]. Consequently, considerable investments were made (and wasted) with little discernment to what was deserving science and what was not. When it was realized that only a very few of the claimed technical solutions had clinical potential, the equally erroneously conclusion was drawn that the technology as a whole had poor prospects instead of realizing that only a very few successful platform technologies can already create considerable value.

With investors tiring of ‘promising’ pre-clinical results and refusing to put more money into RNAi Therapeutics development unless clinical validation was seen, it was up to the science to prove itself in Man. If not, probably all of the significant pure-play RNAi Therapeutics companies, possibly with the exception of Alnylam, would find it impossible to raise capital on acceptable terms. Fortunately, despite the attendant contraction in the number of new development programs, some of the early capital meant that four candidates in particular had entered clinical development in time to be the industry’s chance at unambiguously validating RNAi in Man: ALN-VSP02, Atu027 ALN-TTR01, and ALN-PCS02. If this could be achieved, it was likely that capital would return to the space. If not, the added wait before such clinical validation could come would have been a great setback to the industry, a setback from which it might have been difficult to recover from financially.

ALN-VSP02 and Atu027: clinical safety of two leading delivery technologies

Together with CALAA-01 (sponsor: Arrowhead Research), Alnylam’s ALN-VSP02 and Atu027 by Silence Therapeutics were the three leading RNAi Therapeutics candidates in cancer, with enrolment starting in 2008-2009. Not only were these candidates important in their own right for their medical and commercial potential, they were supposed to clinically validate the three distinct systemic delivery platforms on which they were based: the cyclodextrin-containing polycation RONDEL technology (CALAA-01), the AtuPLEX lipoplex technology (Atu027), and SNALP liposomes (ALN-VSP02). In addition to cancer, together with hepatic applications the commercially most critical application of RNAi Therapeutics in the near to medium term, AtuPLEX has potential for endothelial cell-directed gene knockdown in general, and systemically administered SNALP also for liver and phagocytic cell-directed gene knockdown.

CALAA-01 enjoyed a year head-start and reported a first data update in a high-profile paper in April of 2010 [6]. Attracting the widespread interest was the fact that by taking tumor biopsies, the investigators were able to demonstrate, with the help of the 5’ RACE assay on tumor biopsies, that RNAi had occurred in target tissues. Moreover, evidence was provided that consistent with this functional finding, RONDEL nanoparticles could be detected in the biopsies. On the other hand, the data on the target knockdown was more ambiguous. Although the RNA analysis suggested measurable gene suppression, the protein analysis did not fully support that. The early promise was only short-lived, however, as a subsequent 2010 ASCO presentation (Abstract No: 3022) showed ample innate immune stimulations which forced the company to concede in late 2011 that at least another phase I trial was necessary in an effort to better manage them. In retrospect, it seems obvious that a major omission of the program was in leaving the RNAi trigger (targeting the M2 subunit of ribonucleotide reductase, RRM2) chemically unmodified, meaning that the risk of inducing such responses was quite high. In the absence of evidence for clinical efficacy and unexplained trial delays (it has taken ~3 ½ years to conclude enrolment), the CALAA-01 phase I results were thus unable to positively impact perceptions of RNAi Therapeutics.

An important step forward in that direction was made with the 2011 ASCO presentations of the fully enrolled phase I study of ALN-VSP02 and a quite encouraging interim update for Atu027. The data suggested that Atu027, an endothelial cell-directed multilamellar cationic lipoplex containing an RNAi trigger against PKN3 [7], was surprisingly well tolerated as dose escalation had reached dosages at which gene knockdown efficacy could be expected based on the preclinical animal data. Dose escalation has been ongoing since and has exceeded predicted RNAi-functional doses- and still no dose-limiting toxicities or serious adverse events were claimed as of December 2011. The reason why this is somewhat unexpected is that the positively charged lipoplexes may have been considered prone to induce various innate immune responses [8], also because no immune suppressive regime was used in the trial. It is possible that the extensive 2’-O-methylation of the AtuRNAi-type trigger partly accounted for that. Activations of the alternative complement pathway, however, were noted, although these were claimed to be clinically not significant. Tumor responses by stringent RECIST criteria meanwhile remain to be demonstrated. Nevertheless, the overall safety profile and well-behaved pharmacokinetics have encouraged further investments in the AtuPLEX and related cationic lipoplex delivery platforms from Silence Therapeutics and can be considered a meaningful step forward for the field.

Of the three candidates, the most ambitious phase I study was that for ALN-VSP02, a SNALP formulation that includes two siRNAs, one against VEGF and KSP for anti-angiogenic and anti-proliferative mechanisms of action, respectively. Enrolling 41 patients with advanced solid cancer with liver involvement, this study included a battery of tests, including biopsies to test for target mRNA cleavage and siRNA tissue concentrations, the measurement of tumor blood perfusion as an indicator of anti-VEGF activity, and various other pharmacological parameters. The study succeeded in demonstrating that this SNALP formulation was fairly well tolerated in these advanced cancer patients at dosages of up to 1.0-1.25mg/kg (ASCO 2011 poster #3025). Among the dose-limiting toxicities were a liver failure with subsequent death at 0.7mg/kg, two cases of transient grade 3 thrombocytopenia at 1.25mg/kg, a grade 3 hypokalemia at 1.5mg/kg, and four grade 1-2 rigor/chills at 1.0mg/kg (one case) and 1.25mg/kg (three cases). The death at the 0.7mg/kg dose was deemed to be possibly related to study drug and occurred in a patient where the tumor burden in the liver was quite extensive. The enrolment criteria were subsequently adjusted to exclude similar patients with a greater than 50% tumor burden in the liver.

Based on the pre-clinical studies (AACR 2009 poster #B204), knockdown activity could have been expected at dosages of 1.0-1.25mg/kg. Consistent with this notion, the 5’ RACE for VEGF (but not KSP) RNAi cleavage performed on the biopsies revealed RNAi activity. In terms of functional activity, the Ktrans, a measure of tumor blood perfusion, declined by 40% or more in over half the patients that had received one or more doses and was consistent with anti-angiogenic activity of the drug. It has to be said though that the Ktrans response was not nearly dose related. Evidence for dose-responsiveness, however, was provided when categorizing patients according to RECIST criteria of tumor responses. Accordingly, only one in 13 patients treated with up to 0.4mg/kg had stable disease for two months or more. This compared to 12 of the 24 given higher doses. Moreover, there was a 70% partial tumor response in an endometrial cancer patient at 0.7mg/kg who stayed on study drug for months; at the 1.0mg/kg dose, the recommended dose for further studies, 7 of 11 achieved stable disease. Despite these preliminary signs of activity, there remain questions about the choice of the target genes, particularly the suitability of VEGF as an RNAi target and the absence of detectable KSP cleavage. Moreover, systemic SNALP delivery to even tumors in the liver is thought to require extended blood circulation times in order to harness the EPR effect, yet the half-life of siRNA in the blood was only in the 15-30 minute range. This is consistent with the relatively short C14-PEG lipid anchor in this formulation. Notwithstanding, the ALN-VSP02 study was a stringent test for the safety of SNALP delivery and added considerable clinical pharmacokinetic experience to this technology. Among the latter was the detection of amounts of siRNAs in normal liver which strongly indicated [9] that gene knockdown with SNALP was possible for liver-expressed genes.

ALN-TTR01 and ALN-PCS02: most impressive demonstrations of RNAi in Man

Despite the largely acceptable safety and promising pharmacokinetic data from the Atu027 and ALN-VSP02 studies, the field still lacked black-and-white evidence for target gene knockdown following systemic delivery. The SNALP-enabled TKM-ApoB by Tekmira in early 2010 was close to providing such evidence. Unfortunately, concomitant with the achievement of slight ~20% ApoB reductions, moderate immune stimulations were observed at 0.6mg/kg in the dose escalation trial, causing the company to terminate the trial (http://clinicaltrials.gov/ct2/show/NCT00927459?). The burden thus fell onto ALN-TTR01, another SNALP-enabled RNAi Therapeutics targeting the liver-expressed transthyretin gene. Mutations of this genes frequently cause familial amyloidotic polyneuropathies and cardiomyopathies which shorten the lives of ca. 50,000 patients worldwide.

This time implementing transient immune suppression (corticosteroids and H1/H2 histamine receptor blockade) as a precautionary measure, the trial began enrolling patients in June 2010 in Europe. A little more than a year thereafter, Alnylam presented almost full trial data at the November 2011 FAP conference in Kumamoto, Japan. It came as a great relief that at the highest, 1.0mg/kg dose the 5 patients exhibited a mean reduction of serum TTR of 41% following a single intravenous infusion. One patient exhibited a text-book 81% RNAi-type TTR reduction at nadir (week 1), with pronounced knockdown persisting out to 4 weeks (50%). Without ifs or buts, this was clear demonstration of effective RNAi in Man. Equally important, except for mild-to-moderate infusion reactions which were readily managed by simply slowing the rate of infusion, no meaningful adverse events were reported. As that trial is currently being wrapped up with more patient data expected for the 1.0mg/kg dose cohort, the sponsor Alnylam is aiming to further enhance the competitive profile of its TTR candidate and has filed a CTA for clinical trials with a new, ALN-PCS02-type SNALP formulation for which equivalent gene knockdown can be expected at 10-fold or more reduced dosages (patent application WO 2010/144740 A1). The goal here is to achieve a more potent knockdown with a higher margin of safety and a once every month or two dosing frequency.

The RNAi clinical dataflow culminated in early January 2012 with Alnylam announcing dose escalation data for the phase I study of ALN-PCS02 for the treatment of hypercholesterolemia. As predicted, equivalent knockdown to ALN-TTR01 were obtained at much reduced dosages with this improved ‘MC3-type’ SNALP formulation from Tekmira: ~60% mean peak reductions in serum PCSK9 for the 0.15mg/kg and 0.25mg/kg dose cohorts. As PCSK9 antagonizes ‘bad’ LDL cholesterol removal from circulation [10], its inhibition was accompanied by ~35% reductions in LDL cholesterol. Due to the favorable safety profile (rashes were noted, but these were likely related to route of administration as they also occurred in the placebo cohort), dose escalation is expected to proceed. At the higher dosages, more robust, less variable knockdown can be expected for both PCSK9 and LDL-cholesterol. This would put it in a favorable competitive position vis-à-vis the PCSK9 monoclonal antibody competition (e.g. REGN727/SAR236553).

The most significant limitation of the ALN-PCS02 study turned out to be the use of transient immune suppression. These caused short-lived (+65%) spikes and (-25%) depressions of PCSK9 and LDL-cholesterol, respectively. While transient immune suppression should be acceptable for many of the initial indications of high unmet medical need for which SNALP-based therapeutics are largely being developed at the moment, especially if once every month or two dosing can be achieved, they can not only complicate the analysis of studies such as ALN-PCS02, but would also restrict the eligible patient population for a condition such as hypercholesterolemia. Introduced as a precautionary measure following the TKM-ApoB experience, the question is whether it is possible to do entirely without it as SNALP potency has improved [11], lipid-specific toxicities been minimized, and more predictable innate immune stimulation assays been introduced as presented by Tekmira at a Drug Information Association (DIA) meeting on March 23, 2010, in Bethesda, MD.

Conclusion

The clinical results not only provided the long-awaited clinical validation and are a boost of confidence for the entire RNAi Therapeutics industry, but they directly de-risk two of the most important systemic delivery technologies: SNALP and AtuPLEX. The results with SNALP in particular set the stage for a forceful expansion of this delivery platform with already 5-6 candidates in active clinical development: ALN-VSP02, ALN-TTR01+02, TKM-PLK1, TKM-EBOLA, and ALN-PCS02. More still are expected to enter the clinic over the next two years. The following months should also add to the SNALP clinical experience in the form of results from the fully enrolled and dose-escalated ALN-TTR01 and ALN-PCS02 trials, an update on Tekmira’s cancer therapeutic candidate TKM-PLK1, and safety data from the TKM-EBOLA volunteer study. Since the ASCO 2011 presentation, Atu027 has attracted commercial interest in the form of various technology evaluations of AtuPLEX and related delivery technologies from Silence Therapeutics (partners: InteRNA Technologies, Mirna Therapeutics, an undisclosed Japanese ‘Top Ten’ global pharmaceutical company, and miRagen).

The space, however, awaits confidence expressed in the form of a more major financial commitment by a larger pharmaceutical company. This might break the gridlock caused by Roche’s decision to stop in-house RNAi Therapeutics development, a decision so powerful that it essentially caused all capital to retrench to the sidelines or leave RNAi Therapeutics entirely. What should not be lost is that the current situation also represents an attractive technical risk-financial reward opportunity for those companies that dare rely on their own scientific instincts rather than follow the herd. This includes mid-sized pharmaceutical companies and those in the newly emerged and emerging economies which are playing an increasingly important role in RNAi Therapeutics. As access to capital normalizes, platforms in addition to SNALP and AtuPLEX will likely emerge and help further expand the therapeutic reach of RNAi Therapeutics.

Abbreviations

5’ RACE: 5’ rapid amplification of cDNA ends; Ktrans: volume transfer coefficient; RECIST: Response Evaluation Criteria of Solid Tumors; RONDEL: RNAi/Oligonucleotide Nanoparticle Delivery; siRNA: small interfering RNA; SNALP: stable nucleic acid lipid particle.

References

1. Ledford H: Drug giants turn their backs on RNA interference. Nature 2010, 468: 487.
2. Robbins M, Judge A, Ambegia E, Choi C, Yaworski E, Palmer L, McClintock K, MacLachlan I: Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation. Hum Gene Ther 2008, 19: 991-999.
3. Zamore PD: RNA interference: big applause for silencing in Stockholm. Cell 2006, 127: 1083-1086.
4. Tolentino MJ, Brucker AJ, Fosnot J, Ying GS, Wu IH, Malik, Wan S, Reich SJ: Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization. Retina 2004, 24: 132-138.
5. Aouadi M, Tesz GJ, Nicoloro SM, Wang M, Chouinard M, Soto E, Ostroff GR, Czech MP: Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 2009, 458: 1180-1184.
6. Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas A: Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464: 1067-1070.
7. Aleku M, Schulz P, Keil O, Santel A, Schaeper U, Dieckhoff B, Janke O, Erdruschat J, Durieux B, Roeder N, Löffler K, Lange C, Fechtner M, Möpert K, Fisch G, Dames S, Arnold W, Jochims K, Giese K, Wiedenmann B, Scholz A, Kaufmann J: Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression. Cancer Res 2008, 68: 9788-9798.
8. Yew NS, Scheule RK: Toxicity of Cationic Lipid-DNA Complexes. Adv Genet 2005, 53PA: 189-214.
9. Landesman Y, Syrzikapa N, Cognetta A 3^rd, Zhang X, Bettencourt BR, Kuchimanchi S, Dufault K, Shaikkh S, Gioia M, Akinc A, Hutabarat R, Meyers R: In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR). Silence 2010, 1: 16.
10. Horton JD, Cohen JC, Hobbs HH: Molecular biology of PCSK9: its role in LDL metabolism. Trends Biochem Sci 2007, 32: 71-77.
11. Semple SC, Akinc A, Chen J, Sandhu AP, Mui BL, Cho CK, Sah DW, Stebbing D, Crosley EJ, Yaworski E, Hafez IM, Dorkin JR, Qin J, Lam K, Rajeev KG, Wong KF, Jeffs LB, Nechev L, Eisenhardt ML, Jayaraman M, Kazem M, Maier MA, Srinivasulu M, Weinstein MJ, Chen Q, Alvarez R, Barros SA, De S, Klimuk SK, Borland T, Kosovrasti V, Cantley WL, Tam YK, Manoharan M, Ciufolini MA, Tracy MA, de Fougerolles A, MacLachlan I, Cullis PR, Madden TD, Hope MJ: Rational design of cationic lipids for siRNA delivery. Nat Biotechnol 2010, 28: 172-176.

Alnylam Presents ALN-VSP02 Liver Cancer Data at ASCO 2011

Widely expected, Alnylam presented over the weekend detailed safety and preliminary efficacy data from its multi-dose, dose-escalation phase I trial of ALN-VSP02 in 41 patients of advanced solid tumors with liver involvement. ALN-VSP02 is a SNALP formulation containing 2 siRNAs, one targeting VEGF (anti-angiogenic mechanism), the other kinesin spindle protein (KSP; anti-proliferative).

The data suggest that ALN-VSP02 has an adequate safety profile for such a cancer application at dosages where gene knockdown can be expected with such formulations in normal liver cells. While not geared towards showing efficacy, there are preliminary signs of efficacy with one case of clear tumor response and a dose-dependent increase in the number of stable patients.

Safety profile bodes well for SNALP delivery platform

Probably as, or even more important than studying the safety of ALN-VSP02, this study was the most rigorous test yet for the SNALP delivery platform. The detailed safety data show that while there were some minor toxicities at the lower dosages, innate immune-related toxicities such as chills and rigor become an issue at doses of 1mg/kg and higher, although none of these events were of high severity (grade 3) where they would be considered dose-limiting. There were also two cases of dose-limiting grade 3 thrombocytopenias at the 1.25mg/kg. Overall, this profile justified a recommended dose of 1mg/kg every 2 weeks for future studies.

Importantly, this study strongly suggests that SNALP delivery is amenable to repeat-administration with no apparent changes in the pharmacology upon re-administration. The ability to repeat-administer had been one of the question marks when SNALP delivery was in its infancy and this was the first such clinical experience.

While this safety profile is adequate for most cancer applications and other applications for severe diseases such as Ebola viral infections, the record suggests that for less severe indications, especially when chronic application is required, improvements in the potency and tolerability of SNALP delivery are necessary. Of course, based on Tekmira’s proprietary insights, considerable improvements in the therapeutic index have been achieved since ALN-VSP02 was locked down. In this light, the 1mg/kg mark determined here is very encouraging. Nevertheless, we still have to await clinical data with the newer formulations, starting with ALN-PCS later this year to fully justify such optimism.

Signs of preliminary efficacy

Although not the primary objective of such a study, Alnylam made significant efforts to study whether ALN-VSP02 works as designed and whether there are any signs of anti-tumor efficacy. In this regard, the highlight of the study is probably a patient with primary endometrial cancer and multiple liver metastases that showed a partial, but very considerable 70% tumor response and that has been on study drug for now more than a year. Moreover, among evaluable patients, only 1 in 13 given a dose up to 0.4mg/kg had stable disease for 2 months compared to 12 in 24 dosed with larger amounts of study drug (caveat: base-line criteria may not be fully comparable as the patient recruitment criteria were slightly adjusted during dose escalation).

In terms of mechanism of action, 5’ RACE RNAi cleavage results and a reduction in tumor blood flow and leakiness as measured by DCE-MRI support an anti-angiogenic mechanism of action due to VEGF knockdown, but overall the mechanism of action data fell slightly short of my expectations, although technical challenges in taking such biopsies and analyzing them may account for that. In particular, no data supporting a knockdown of KSP was reported. 5’ RACE cleavage assays apparently suffered from technical challenges, and the analysis of mitotic spreads was not mentioned at all.

Study validates SNALP siRNA delivery to liver

When I reviewed last year ALN-VSP02, I was a bit confused about Alnylam’s choice of a short-circulating C14-PEG-lipid instead of a longer-circulating C18-PEG-lipid. I even thought that there might be a mistake in Alnylam’s cartoons of the formulations, but my question remained unanswered. Having listened to the recent ThinkEquity presentation by Tekmira’s CEO Mark Murray, it is clear to me that this was not a typo: Tekmira’s solid cancer candidate TKM-PLK1 is a longer-circulating formulation, and as we know, Tekmira is getting more and more interested in competing with ALN-VSP02 in liver cancer.

Why does C14 vs C18 matter? In short, C14-PEG-lipids are most suited for gene knockdown in normal liver cells, not liver cancer cells. Normal hepatocytes largely derive their blood supply from the portal vein, whereas most liver cancers primarily tap into the arterial blood supply. For this reason, even when addressing tumors in the liver, a more stable formulation would have been desirable. This also applies to tumors outside the liver (note: the vast majority of patients in Alnylam’s phase I study had such turmors).

Therefore, when Alnylam reports siRNA concentrations in the liver biopsies that were often well above concentrations associated with potent knockdown of genes expressed in the liver in pre-clinical studies, it is mainly a validation that the DLinDMA C14 formulations successfully deliver siRNAs to the liver.

Why did Alnylam choose C14? This may be more of a rhetorical questions and if you follow the Tekmira litigation you will understand why. In other words, my suspicion is that if Alnylam were to develop another liver cancer candidate, it would indeed choose a longer-circulating formulation.

In conclusion, the ALN-VSP02 phase I study nicely adds to the accumulating evidence that up to 1mg/kg of SNALP-siRNA is readily feasible which predicts a comfortable therapeutic index for the liver-targeted SNALP pipeline. ALN-VSP02 will likely have to be studied in more targeted patient population and together with other therapeutic modalities before its real potential becomes clear. It won’t be an easy stroll, but the data show that there are a few reasons to be optimistic.

PS: Silence Therapeutics also presented today at ASCO an update on Atu027 for advanced solid cancers. The press release can be found here. I am currently traveling and will provide further commentary when I get a chance.