Tag Archives: clinical trial

Honoring a fallen hero in the struggle against Cystic Fibrosis and AR extremism

Yesterday I learned some sad news via the Understanding Animal Research blog, that a young woman named Laura Cowell had died, succumbing to cystic fibrosis at the age of just 25.  To see a life so full of promise end so prematurely is always sad, but what makes this death so gutting is that Laura is one medical research’s heroes. I never met Laura, but back in the bad old days a decade ago – when the animal rights extremist campaign against medical research in the UK was at its height – she had the courage to stand up and voice public support for the animal research that is so crucial to progress against diseases such as cystic fibrosis.  For this Laura and her mother Vicky, who chaired the now-disbanded (after a job well done!) patient advocacy group Seriously Ill for Medical Research, have my unwavering respect and admiration.

Laura Cowell – a brave campaigner for cystic fibrosis research

In an article in the Times yesterday science correspondent Mark Henderson wrote about Laura’s bravery:

Most researchers who worked with animals were reluctant to fight back. Their fears were far from unreasonable: Professor Colin Blakemore, one of the few to have done so, was repaid with letter bombs addressed to his children. Politicians deplored the threats while doing nothing about them. Cravenly, the governing Labour Party dropped HLS shares from its pension fund portfolio, and Blakemore was blackballed for a knighthood because of his “controversial” stance on vivisection.

It was against this background that a 16-year-old girl decided to speak out. Laura Cowell was born with cystic fibrosis, and took 40 pills a day to keep her illness and its complications at bay. “I rattle,” she used to joke. Animal research, however, she took seriously. Drugs developed through vivisection were keeping her alive. In 2002, she agreed to front a campaign that aimed to explain the benefits of animal experiments, as living proof of their contribution to medicine. ”

As Mark points out it was the willingness of people like Laura Cowell and Colin Blakemore to speak out, despite the threat from extremists and petty insults from more mainstream animal rights groups, in favour of animal research that turned the tide of public opinion in the UK favour of animal research, culmination in Laurie Pycroft’s stand against animal rights extremists in Oxford and the founding of Pro-Test.

Ipsos-MORI polls show unconditional support for animal research has almost doubled since 1999, and growing trust in the regulations that govern it. In 2005, laws against harassment were introduced and a police extremism unit was tasked with targeting violent activists. As ringleaders were jailed, the intimidation stopped. The Oxford lab was built. A climate of fear no longer threatens an important branch of British science. ”

Mark then goes on to criticize the failure of some medical research charities to respond to a recent attempt by the animal rights group Animal Aid to persuade people to stop donating to medical research charities (at a time of declining income for many charities because of the recession) that support animal research, which focused on the British Heart Foundation (BHF), Cancer Research UK (CRUK), the Alzheimer’s Society and Parkinson’s UK. I’m not sure that Mark is being fair, the BHF (who also highlight animal research in a new campaign), CRUK and Parkinson’s UK all issued strong statements explaining the value of animal research to their work, and while the Alzheimer’s Society didn’t issue a specific statement on this occasion they frequently discuss animal research in their research news and in 2010 wrote in a position statement that:

However, the Alzheimer’s Society and its trustees believe that funding medical research with animals remains essential if we are ultimately going to understand the causes of dementia and develop effective treatments.”

It is clear that medical charities in the UK are increasingly prepared to stand up for the importance of animal research to medical progress, something that is very refreshing to those of us who remember how things were a decade ago, and as Mark points out medical charities are uniquely well placed to act as advocates for animal research due to the respect and admiration that the public have for their work. For this sea change in attitudes towards animal research in the UK we have to thank the courage of individuals like Laura Cowell.

So how should we honor Laura?

We have discussed the contribution of animal research to the development of existing therapies – and future cures – for cystic fibrosis research on the Speaking of Research Blog in a couple of occasions, including the promising research being undertaken by the UK Cystic Fibrosis Gene Therapy Consortium. The consortium is planning to launch in 2012 the first ever clinical trial to examine if gene therapy can improve lung function in people with cystic fibrosis, using a vector whose development relied on information obtained from studies in mice, and which has already had promising results in a pilot study in CF patients. This clinical trial is being supported by the Cystic Fibrosis Trust who recently launched a fundraising campaign to raise the £6 million required to pay for the trial.

Laura Cowell died before gene therapy for cystic fibrosis could become a reality in the clinic, but there are many cystic fibrosis patients alive today – and many more yet to be born – who may in future benefit from it.

So I invite you to remember Laura by making a donation to the Cystic Fibrosis Trust – Gene Therapy fund.

Paul Browne

The Value of Animals in Pre-Clinical Trials

In the early stages of development a new drug must be tested in a series of human clinical trials.  The earliest phases of trials aim to assess the safety and tolerance of a drug in human volunteers.  In planning such trials you face an obvious question: what initial dosage to use? Of course, you want to do your best to avoid giving human volunteers drugs at dosages that may be harmful to them.  So you ask yourself — Is there any way to get some information about safety limits before proceeding with exploratory human studies?  Specifically, could data in pre-clinical studies where we expose cell cultures or animals to the drug be used to predict the maximum human tolerance dosage for the drug?
In one classic study, Freireich et al (1966) performed a quantitative comparison between the toxicity of anti-cancer agents in mouse (and other species such as rats, dogs, and monkey) and the human maximum tolerated dose.  All species provided good predictability of human response, but let us look in detail as some data from the mouse.
These investigators measured the dosage (in milligrams per surface area) at which 10% of the mouse population died (so-called lethal-dose 10%, or LD10) and compared that to the maximum tolerance showed by human volunteers.  The comparison was done across 18 different anti-cancer drugs.  The data are re-plotted below with the x-axis representing the LD10 value in mice and the y-axis the maximum tolerated dosage in humans. The actual data points are the open blue circles and the red line represents the best linear fit.
The correlation coefficient — a measure of association between the mouse and human data —  is 0.95.  A perfect agreement between mouse and human response would have resulted in value of 1.0.  If there was no relationship whatsoever between the two datasets, as claimed by some opponents of animal research, the correlation coefficient would have been near zero. As it turns out, 90% of the variability in the human data is accounted for the mouse data.  The likelihood of such figure resulting by chance is less than 1 in a billion.  In other words, the association between the mouse and human data is highly significant.
To illustrate the meaning of these numbers consider the following scenario.  Assume you are in the possession of this graph and you are asked to volunteer to participate in a clinical trial for a new potential anti-cancer drug which has an LD10 value in mice of 10 mg/m2.  Would you be willing accept an initial drug dose of 100 mg/m2?  What if the LD10 value of the proposed drug was instead 10000 mg/mm?  Would you now be more or less willing to accept participation in a trial with initial drug dose of 100 mg/mm2?
Was the graph useful to you in making a decision about your participation on this clinical trial?  Would you rather make your decision based on such data or by flipping a coin?  Do you honestly believe the two methods equivalent?  Don’t you think it would be important for scientists to obtain such information before proceeding to inject arbitrary doses of a drug in human volunteers?
True, toxicology assessments are not perfect and they can be improved.  In fact the methods for the evaluation of novel cancer therapeutics have indeed been improved over the past decades.  There is also no doubt that, in this process, we are morally obliged to seek alternatives to the use of animals in drug screening and testing, and work in this area is actively pursued.  If such methods can be developed and validated they should be adopted.  It should be noted, however, the use of animals in toxicology is arguably one of the least common uses of animals in science.  Attempts to equate toxicology testing to the whole enterprise of animal research make no sense at all.
Anyone confronted with the above evidence that continues to argue that in going into a clinical trial “you might as well flip a coin” is simply not credible: pre-clinical trials in cell cultures and animals have predictive power.
Anyone claiming to be in a possession of a theory that proves animals cannot be predictive of human response is wrong — as this position is refuted by the data.
Anyone denying the uncountable contributions of animal research to human health is intellectually dishonest or ignorant of medical history.
Animal rights activists are free to object about the use of animals in research based on ethical grounds, but they cannot do so by robbing science of its accomplishments or denying the important role they play in medical science.

Microbicide gel cuts HIV infection rates…thank the monkeys!

There was exciting news on Monday when it was announced at an international AIDS conference in Vienna that microbicide gel had dramatically reduced the transmission of HIV in a Phase 2 clinical trial involving 889 women in South Africa.  If confirmed by  larger phase 3 trials this gel will offer millions of women a way to protect themselves against this dread disease that blights communities around the world.

Dr Abdool Karim explains how to use a microbicide gel applicator. Image courtesy of CAPRISA.

Unlike previous microbicide gels that failed to offer significant protection against HIV infection this gel included the anti-retroviral drug tenofovir. Regular readers of this blog may recognize tenofovir, it was discussed in an article on the role of non-human primate research in developing HIV prophylaxis by virologist Dr. Koen Van Rompay that we posted last year.  Dr. Van Rompay’s article looked at the use of oral tenofovir in pre- and post-exposure prophylaxis rather than its use in a microbicide gel.

So did the research on preventing SIV transmission in monkeys influence the decision to use tenofovir in this microbicide gel? You betcha! In the first report of a Phase 1 trial of this tenofovir-containing microbicide gel published in 2006 (1) the authors state that the success of tenofovir in preventing SIV infection on monkeys – the same research discussed by Dr. Van Rompay – was a deciding factor when they took this gel into clinical trials.

‘Tenofovir gel, 9-[(R)-9-(2-phosphonylmethoxyprophyl) propyl]adenine monohydrate, a nucleotide reverse transcriptase inhibitor, has demonstrated ability to inhibit retroviral replication in animals and humans, and it has been well tolerated when used orally, as tenofovir disoproxil fumarate, (tenofovir DF; Viread) [16–20]. Tenofovir DF has been approved for treatment of HIV-1 infection and is increasingly used as part of therapeutic regimens for HIV-positive individuals [21]. Tenofovir has been proven to be effective in blocking the transmission of SIV in animal models when given as pre- or postexposure prophylaxis systemically or when applied as an intravaginal gel [22–25]. Tenofovir bisphosphate, the active intracellular moiety, has a very long intracellular half-life (> 72 h), which could allow for more convenient, coitally independent intravaginal use [26]. Given the data showing animal protection with tenofovir gel, and the extensive human safety data with oral tenofovir in HIV-positive patients, the HIV Prevention Trials Network (HPTN) decided to assess the safety and tolerability of tenofovir gel in HIV-negative and HIV-positive women and their male sexual partners (HPTN 050).’

The above passage also mentions that they tested whether the microbicide gel containing tenofovir could prevent vaginal SIV transmission in monkeys*, and the finding that it could drove their subsequent decision to take the gel into clinical trials.  This was an important decision, a review of HIV microbicide gels published in the journal Science (2) two years ago pointed out the failure to evaluate other microbicide gels in monkey models of HIV transmission allowed these gels to proceed into clinical trials where they subsequently failed.  It is notable that the microbicide PRO 2000, also evaluated in monkeys, is the only other microbicide to demonstrate an ability (albeit less dramatic) to prevent HIV infection in clinical trials.

So what now? Well the tenofovir containing gel will go on into larger phase 3 trials to further evaluate its ability to prevent HIV infection in women. In the meantime following a study showing that it can prevent the transmission of rectal SIV transmission in macaques (3) this gel is now in phase 1 safety trials in men.

This is welcome news after years of disappointment, and further evidence that where HIV is concerned there can be no shortcuts; all therapies whether microbicide gels or vaccines must be thoroughly evaluated in stringent animal models before being taken to human clinical trials. Perhaps now we can start to turn realism into optimism.

In other news this week, Americans for Medical progress have announced the 2010 Michael D. Hayre Fellows in Public Outreach. Neuroscientists Elizabeth Burnett and Scott Dobrin will use the fellowship grant to develop their project “Speaking Honestly – Animal Research Education (SHARE)”, which is designed to guide educators in leading classroom discussions on the humane use of animals in research in an engaging and interactive manner. We wish them the very best as they follow in the footsteps of the first Hayre fellow, Speaking of Research founder Tom Holder.

* Unfortunately this study was never published in the scientific literature, this is something that sometimes happens with pre-clinical studies performed by biotechnology and pharmaceutical companies…usually because they wish to keep the work confidential for commercial reasons…and is a source of great frustration to people like me who write about this work!

Paul Browne

1)      Mayer K.H. et al. “Safety and tolerability of tenofovir vaginal gel in abstinent and sexually active HIV-infected and uninfected women.” AIDS. volume 20(4), pages 543-551 (2006), DOI:10.1097/01.aids.0000210608.70762.c3.

2)      Grant R.M. “Whither or wither microbicides?”  Science. Volume 321(5888), pages 532-534 (2008), PubMed Central:PMC2835691.

3)      Cranage M. et al. “Prevention of SIV Rectal Transmission and Priming of T Cell Responses in Macaques after Local Pre-exposure Application of Tenofovir Gel” PLoS Med. Volume 5(8):e157(2008) DOI:10.1371/journal.pmed.0050157

RNAi: Send in the Nanobots!

The publication of the preliminary results of a small clinical trial of a new therapy called RNA interference (RNAi) online in the scientific journal Nature is causing quite a stir in the scientific community this week.  A team led by Professor Mark E. Davis at Caltech targeted the delivery of a nanoparticle only 70 nanometers in diameter containing small interfering RNA (siRNA) to cancer cells in three patients with metastatic melanoma, which reduced the levels of a protein called RRM2 that is required for the tumour growth.  This trial is the result of over a decade of research in organisms as diverse as nematode worms, mice and monkeys, but why is the result of this trial so noteworthy? And what is RNAi anyway?

Cancer genes in human melanomas have been switched off. Image courtesy of the National Cancer Institute

If you have ever studied biology you will probably be familiar with the “central dogma of molecular biology”; it describes how our genes encode the proteins that are the building blocks, and indeed the builders, of all the cells in our bodies. The very short version is that our genes are made up of sequences of double stranded DNA consisting of the deoxyribonucleotides A,C, G and T, and these sequences are transcribed by a protein called RNA polymerase into matching sequences of the single stranded messenger RNA (mRNA) , made from the ribonucleotides A, C, G and U. Another protein complex known as the ribosome then translates the mRNA sequence into a corresponding sequence of amino acids that when completed make up a brand new protein.  Our new protein almost invariable undergoes further processing but we needn’t concern ourselves with that here.  RNAi is the process where an assembly of proteins named the  RNA-induced silencing complex (RISC) binds short double stranded segments of RNA that in turn target RISC to particular mRNA sequences to which they are complementary.  RISC breaks down the mRNA molecule, preventing production of its associated protein and effectively silencing the targeted gene. The beauty of RNAi is that it allows an organism to target specific mRNA molecules for destruction, and it is a mechanism for regulating the flow of genetic information whose importance we are still only beginning to appreciate.

RNAi was discovered only 12 years ago by Andrew Fire and Craig Mello through their basic research on the regulation of gene expression in the nematode worm Caenorhabditis elegans, a discovery which earned then the Nobel Prize in 2006. C.elegans is a popular model organism for scientists studying gene function and development, its small size and simple structure make it relatively easy to follow the fate of individual cells, while as an animal it shares many of its genes and biological processes with mammals.  This turned out to be the case with when in 2001 it was shown that RNAi helps mice to control hepatitis B infection, and scientists began to examine whether RNAi could be used therapeutically (1). To do this scientists made siRNA, an artificial version of the short double stranded segments of RNA that target RISC to complementary mRNA sequences, and early experiments in mice demonstrated that siRNA induced RNAi could reduce the levels of target proteins in mice.  The first human trials of RNAi began in 2004 for the treatment of wet age related macular degeneration and at first seemed promising, but suffered a setback when further research in mice revealed that the “naked” siRNA injected into the eye in these trials actually stimulated an immune response that was responsible for at least some of the benefits seen in earlier trials (2). This was a worry as an unwanted immune response might lead to an adverse reaction if the siRNA was injected into the bloodstream rather than a small part of the eye.

In recent years scientists have been developing technologies that allow injected siRNA to evade the immune system and target only those tissues where RNAi activity is desired,  reducing the quantity of siRNA that needs to be injected and also the risk of adverse  effects due to RNAi affecting off-target tissues. Mark E Davis, a professor of chemical engineering at Caltech and one of the scientists leading these efforts, uses polymers that assemble with siRNA to form a nanoparticle that resembles a tiny ball with siRNA at its centre.  The nanoparticle shell protects the siRNA from being broken down while it is circulating in the bloodstream, and then interacts with the cell membrane to help the siRNA enter a cell so that it can do its job.  Of course he didn’t want the nanoparticle to release its siRNA payload into any old cell so he attached a protein called transferrin as a targeting ligand to the nanoparticle. Tumour cells express far more of the transferrin receptor on their surfaces than normal cells, and the hope was that the nanoparticles would bind to tumour cells in preference to normal cells.  To test whether this would work Prof. Davis team injected the nanoparticles, containing a siRNA that targeted a cancer gene, into mice that had metastatic Ewing’s sarcoma(3). They observed that the transferrin labelled nanoparticle delivered the siRNA to the tumour cells, knocked down the activity of the target cancer gene and dramatically slowed tumour growth, and when the transferring ligand was removed his effect not seen.  They also observed that the nanoparticle did not stimulate the immune system or affect any of the major organs of the mouse, indicating that their method had solved safety problems seen in earlier RNAi trials.

The targeted nanoparticle used in the study and shown in this schematic is made of a unique polymer and can make its way to human tumor cells in a dose-dependent fashion. Image courtesy of Derek Bartlett and the California Institute of Technology.

Prof. Davis and his colleagues next needed to identify an appropriate target for human trials of their nanoparticle siRNA delivery system, and decided to target the M2 subunit of Ribonuclease reductase (RRM2), a protein that is required for cell division and which has recently been the subject of a lot of research as a target for anti-cancer drugs.   They first used in vitro studies to identify a siRNA sequence that effectively targeted the RRM2 mRNA, which they named siR2B+5, and then demonstrated in mice that this siRNA could block the production of RRM2 and reduce the growth of tumours (4).  As a final safety evaluation prior to human trials they injected different doses of their nanoparticle  containing siR2B+5 and labelled with transferrin to cynomologus monkeys, whose RRM2 mRNA is targeted by siR2B+5 in exactly the same way as in humans,  and found that it was safe and did not produce any unwanted effect on the immune system (5).

The human clinical trial reported this week confirmed that transferrin-labelled nanoparticle injected into the bloodstream were safely delivered siR2B+5 to the tumours of metastatic melanoma patients, and that the siRNA knocked down the production of RRM2 protein by RNAi (6).  Of course this is only a preliminary result, at this stage we don’t know to what extent this experimental treatment will reduce tumour growth in these patients, let alone if it will cure their cancer. If it is a success it will probably need to be combined with other anti-cancer drugs to be fully effective, so it is good to know that thanks to animal research other nanotechnology based drugs such as Lipoplatin are in clinical trials that offer more potent anti-cancer activity with less toxicity than existing anti-cancer drugs. Nonetheless to focus on this uncertainty would be to miss why this small trial is causing such excitement; for the first time scientists have shown that it is possible to target RNAi therapy to a particular tissue type within the body, and that is a breakthrough that opens up a whole new area of medicine. The era of RNAi medicine has begun!

Paul Browne

1)      McCaffrey A.P., et al. “RNA interference in adult mice.” Nature Volume 418, pages 38–39 (2002) DOI: 10.1038/418038a

2)      Castnotto D. and Rossi J.R. “The promises and pitfalls of RNA-interference-based therapeutics” Nature Volume 457(7228), pages 426-433 (2009) DOI:10.1038/nature07758

3)      Hu-Lieskovan S. et al. “Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma.” Cancer Re. Volume 65 (19), Pages 8984-8992 (2005) DOI:10.1158/0008-5472.CAN-05-0565

4)      Heidel J. et al. “Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo” Clin. Cancer Res. Volume 13(7), Pages 2207-2215 (2007) DOI: 10.1158/1078-0432.CCR-06-2218

5)      Heidel J. et al. “Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA.” Proc.Natl Acad. Sci. USA Volume 104(14), Pages 5715-5721 (2007) DOI: 10.1073/pnas.0701458104

6)      Davis M.E. “Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles”  Nature Advance Online Publication 21 March 2010 DOI:10.1038/nature08956

Protecting a broken heart: the discovery of remote ischemic preconditioning.

After a couple of weeks dominated by dialogue with moderate animal rights activists, and subsequently the response of the scientific community to threats by animal rights extremists,  it is refreshing to be able to turn again to an example of how research on rabbits and dogs is furthering medical progress.

The prospects of surviving a heart attack have improved greatly over the past few decades, and thanks  to the development of surgical techniques such as coronary artery bypass and clot-busting thrombolytic drugs many patients go on to live long and healthy lives who would previously have faced an early grave.  Despite this progress doctors and scientists are still looking for ways to further reduce the toll of death and infirmity that results from heart attacks; now a report on the BBC suggests that another important advance is in progress.

Figure A is an overview of a heart and coronary artery showing damage (dead heart muscle) caused by a heart attack. Figure B is a cross-section of the coronary artery with plaque buildup and a blood clot. Image displayed courtesy of the National Heart, Lung and Blood Institute.

A team led by Professor Hans Botker of Aarhus University Hospital in Denmark reported a clinical trial of over 300 patients where a novel technique known as remote ischaemic preconditioning (rIPC) safely reduced the amount of damage suffered by the heart during ischemia, when its blood and oxygen supply is cut off during a heart attack (1).  rIPC is a phenomenon whereby short periods of ischemia in one tissue can protect a distant tissue or organ from longer periods of ischemia. In this trial the blood supply to muscles in the arm was cut off using a blood pressure cuff for brief periods in heart attack victims on their journey to hospital, and it was used in addition to established treatments.

So how does it work? Well the answer is that we still don’t know. Research in animals indicates that the tissue exposed to brief periods of ischemia release factors that then travel through the bloodstream to other organs where they alter the metabolism in that organ to make it more resistant to damage from oxygen starvation, but the identity of these factors had not yet been confirmed (2).  This raises an obvious question, if the mechanism is so poorly understood how was this phenomenon identified? After all without this knowledge  in vitro or computational studies could not have identified it, and doctors could hardly go around stopping the blood flow in the arms of heart attack victims without having a very good reason for doing so!

This story starts in the mid 1980’s when scientists studying heart attacks in dogs observed that while blocking a major coronary artery for an extended period resulted in the same damage seen in heart attacks in humans, brief blockage of blood flow did not result in this damage, even if repeated several times.  In fact they observed that the energy use in the heart was slower in later periods of transient ischemia than in the first period, reducing its need for oxygen, and postulated that multiple brief periods of ischemia in the heart might prevent it from damage in a subsequent longer period of ischemia. When they tested this in dogs they found that was indeed the case, four 5 minute periods of ischemia did indeed reduce the heart damage seen after a sustained 40 minute period of ischemia (3).  Subsequent experiments confirmed this finding, and in later clinical trials the technique was found to be beneficial for patients undergoing heart surgery where the supply of blood to the heart is cut off.  Despite this utility the technique of directly preconditioning the heart has been restricted to situations where it is possible to operate on the patient before the supply of blood to the heart muscle is cut off for a prolonged period, and it is not a viable option with heart attack victims.

At this point further analysis of the studies undertaken in dogs suggested a way to widen the clinical use of this technique, as it was noticed that preconditioning one area of heart tissue protected other areas from subsequent damage. Might it be possible to protect the heart by inducing transient ischemia in other tissues? Initial studies in animals and subsequent human trials examined transient ischemia of the mesentery and kidney, discovering that it could reduce damage to the heart. However inducing transient ischemia in the mesentery and kidney still required surgery and was hardly ideal for emergency situations. The breakthrough came with the demonstration by Yochai Birnbaum and colleagues at the Good Samaritan Hospital that inducing transient skeletal muscle ischemia in a rabbit model of heart attack substantially reduced the damage to the heart (4), a result subsequently confirmed by other scientists studying heart attack in rats and rabbits.  The significance of this discovery is that it is possible to block the blood flow to skeletal muscle through the use of a standard blood-pressure cuff, avoiding the necessity for additional surgery.

Thanks to pioneering work of Yochai Birnbaum and other animal researchers successful clinical trials of the blood pressure cuff to induce transient ischemia in limb muscles have been reported in children undergoing heart surgery (5) and now in heart attack victims.  We hope that in years to come this exciting new technique will fulfill its early promise and help save many lives.

Paul Browne, PhD

1)      Botker H. E. et al. “Remote ischemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomized trail” The Lancet Volume 375 (9716), Pages 727-734 (2010) DOI:10.1016/S0140-6736(09)62001-8

2)      Shimizu M. et al. “Transient limb ischemia remotely preconditions through a humoral mechanism acting directly on the myocardium: evidence suggesting cross-species protection” Clinical Science, Volume 117, Pages 191-200 (2009) DOI:10.1042/CS20080523

3)       Murry C.E., Jennings R.B., Reimer K.A. “Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium.” Circulation Vol.74(5), Pages 1124-1136 (1986) PMID: 3769170

4)      Birnbaum Y., Hale S.L. Kloner R.A. “Ischemic preconditioning at a distance: reduction of myocardial infarct size by partial reduction of blood supply combined with rapid stimulation of the gastrocnemius muscle in the rabbit.” Circulation Vol. 96(5), Pages 1641-1646 (1997) PMID: 9315559

5)      Cheung M.M. et al. Randomized controlled trial of the effects of remote ischemic preconditioning on children undergoing cardiac surgery: first application in humans” J. Am. Coll. Cardiol. Volume 47(11), Pages 2277-2282 (2006) doi:10.1016/j.jacc.2006.01.066