Pro-Test Blogs!

Heart failure, where the heart is unable to maintain a sufficient blood flow to supply the body’s needs, is a leading cause of death, especially among the over 65’s. Half of all chronic heart failure patients die within four years of diagnosis. It can have a number of causes, for example damage to heart tissue after a heart attack, and leads to a variety of problems in patients. Fatigue and muscle weakness are common as the muscles receive insufficient oxygen, and because waste products cannot be removed from tissues quickly enough fluid can build up in the lungs and other parts of the body, often the legs and abdomen. The extra strain placed on the heart as it tries to maintain adequate blood pressure can lead to further damage to the heart and ultimately cardiac arrest.

In heart failure the rate at which the heart beats is often increased, and group of scientists led by Karl Svedberg and Michael Komajda set up the SHI_fT study, to evaluate whether a drug called Ivabradine, which lowers the heart rate, could reduce risk of death or hospitalization in a group of patients who had heart failure accompanied by an elevated resting heart rate. Significantly fewer patients taking Ivabradine in addition to their existing treatments required hospital admission during the course of the study, compared to a control group who were given a placebo in addition to their existing treatment. The most striking outcome was that Ivabradine cut the risk of death by 26%.

So what is Ivabradine, and where does it come from?

Ivabradine slows the heart rate by inhibiting an electrical current known as the I_f current* which is a major regulator of the activity of the sinoatrial node – better known as the pacemaker. Inhibiting the I_f current slows the generation of the electrical impulses by the sinoatrial node that trigger heart contraction, and therefore slows the heart rate itself. Ivabradine, then known as S16257, was first developed in the early 1990’s when it was found to be able to block the I_f current in-vitro in sinoatrial node tissue from rabbits and guinea pigs, and slowed the generation of electrical impulses in a manner that was safer than other bradycardic drugs (1). Ivabradine was then evaluated in live rats and dogs, where it safely reduced the heart rate, and moreover did so without reducing the blood pressure (2,3). While beta-blockers such as Propranolol can reduce the heart rate they also lower the blood pressure – indeed they are used to treat hypertension - and hence are not suitable for many patients, so the development of a drug that could reduce heart rate without affecting blood pressure was very welcome.

Following the successful animal studies Ivabradine entered human clinical trials and in 2005 was approved for the treatment of angina pectoris. In angina pectoris the heart muscle receives too little oxygen, a problem exacerbated by a fast heart beat that increases the need for oxygen, so lowering of the heart rate by Ivabradine reduced oxygen demand and prevents angina attacks. The success of Ivabradine in the treatment of angina pectoris in turn led to its evaluation in heart failure.

The successful outcome of SHI_fT study is a major boost to the development of better treatment regimes for heart failure, and if it is confirmed by further clinical trials will improve and prolong the lives of many heart failure patients.

* Hence the name of the SHI_fT study - Systolic Heart failure treatment with the I_f inhibitor ivabradine Trial

Paul Browne

1) Thollon C. et al. "Electrophysiological effects of S 16257, a novel sino-atrial node modulator, on rabbit and guinea-pig cardiac preparations: comparison with UL-FS 49." Br J Pharmacol. Volume 112(1), Pages 37-42 (1994) PubMedCentral:PMC1910295

2) Gardiner S.M. et al. "Acute and chronic cardiac and regional haemodynamic effects of the novel bradycardic agent, S16257, in conscious rats." Br J Pharmacol. Volume 115(4):579-586 (1995) PubMedCentral:PMC1908496

3) Simon L. et al. "Coronary and hemodynamic effects of S 16257, a new bradycardic agent, in resting and exercising conscious dogs." J Pharmacol Exp Ther. Volume 275(2), Pages 659-666 (1995) PubMed:7473152

It’s just over a decade since the completion of the first working draft of the human genome was announced, and seven years since the publication of the complete sequence, but in that short time the impact of this new knowledge on all areas medical research has been immense. Sequencing the human genome was a huge achievement, but having got the sequence an even greater task confronts scientists - working out what it all means. To do this scientists have studied the natural variations that exist between individuals, and have also sequenced the genomes of a wide variety of species, some closely related to us, others separated from us by hundreds of millions of years of evolution. Scientists can analyze the similarities and differences between the genes of different species, and examine how changes to the structure or regulation of these genes are reflected in physiology. In many cases it is also possible to use genetic modification to study the function of conserved genes in other species in ways that are just not possible, for technical and/or ethical reasons, in humans. A study published a couple of weeks ago in the scientific journal Nature provides an excellent example of how animal research contributes to our understanding of the human genome.

As the cost of technology such as DNA microarrays has fallen genome-wide association studies (GWAS) have become an increasingly popular way of examining the relationship between genetic differences between individuals and particular diseases. In a GWAS the whole genome of many individuals is screened for variations, and then any association between those variations and particular phenotypes or diseases is determined. Tanya M. Teslovich and colleagues (1) analysed the genomes of over 100,000 people who had been enrolled in 46 separate clinical studies, and identified 95 genes that have variants associated with increases in blood lipid (fat and cholesterol) levels. One of the problems with GWAS studies is that while they are often good at identifying genes that are associated with a disease, they are not so good at identifying which genetic variations actually cause disease, or explaining how the genetic variations contribute to disease. This is where Tanya Teslovich and colleagues scored highly; they were able to show that 14 of the 95 lipid-associated genes were also associated with the development of coronary artery disease, supporting the proposition that elevated blood lipids contribute to coronary artery disease. They also found that overall the effect of the variants was additive, the more risk variants of these 95 genes you have the greater your chance of having elevated blood lipids.

So that established that the gene variants were associated with elevated blood lipids, but to use that information to develop new treatments you need to know how the particular gene affects lipid levels. As you might expect many of the 95 genes identified were already known from previous studies to be involved in the regulation of blood lipids, and in several cases their precise role has been thoroughly studied. However, several of the genes had not been implicated in regulating blood lipids before, and the team decided to use genetically modified mice to investigate their function. They injected viral vectors into the liver of the mice that contained either an extra copy of the gene being studied, to increase expression of the gene, or a short-hairpin RNA, to target the gene for knockdown via RNAi. This allowed them to discover that one gene, GALNT2, decreases levels of high-density lipoprotein cholesterol (HDLC), the so-called “good cholesterol”, while two other genes, Ttc39b and Ppp1r3b, increase HDLC.

Another associated paper (2) in the same issue of Nature takes the analysis even further. Several studies, including the GWAS performed by Tanya M. Teslovich and colleagues, had demonstrated that variations in a particular region of chromosome 1 known as 1p13 were associated with high levels of Low-density lipoprotein “bad” cholesterol (LDLC) in the blood and heart disease, but that these variations were not within the coding sequence of any genes, so they would not affect the structure of any proteins. They first show through genetic studies of human subjects and human liver tissue culture that variations at 1p13 affect the expression of several genes – and hence the amount of protein produced by those genes - and that one particular variation creates a binding site for the transcription factor C/EBP. Transcription factors are proteins that regulate the expression of genes, and this particular site altered the levels of a gene named SORT1. But what does SORT1 do? To answer this they again turned to GM mice, using virus vectors that specifically reduced or increased the levels of SORT1 in the mouse liver. Reducing or eliminating SORT1 expression in the mouse liver led to a reduction in the levels of LDLC in the blood, and that this was found to be due to SORT1 regulating the production of very-low-density lipoprotein (VLDL), a precursor to low-density lipoprotein, in the liver. As a result of this work a whole new pathway for the regulation of blood lipids has been uncovered, one that may offer new opportunities to scientists developing treatments for hypercholesterolemia.

As a BBC news report indicates, the identification of these genes and the elucidation of their function may aid the development of better diagnostic tools to identify those at risk of heart disease, and ultimately the development of better treatments.

These studies illustrate how important animal models, particularly GM mice, are to efforts to decode the human genome. As the biosciences move towards a more systems based approach to biology, one where knowledge of how networks of genes interact to produce a particular physiological or clinical outcome is applied to areas such as toxicology, the information that studies of GM animals can yield will become increasingly important. This importance has not gone unrecognized by the wider scientific community, the 2007 Nobel Prize in Medicine was awarded to Mario Capecchi, Sir Martin Evans, and Oliver Smithies for their discoveries of " principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

With this in mind let’s turn briefly to another GM animal that’s been in the news lately - the rat. While GM mice have become a mainstay of modern scientific research the rat has lagged behind, which is a shame since the larger size, longer lifespan, and more complex behavior of rats make them more effective animal models than mice for studying many human diseases, particularly neurological conditions. The lack of GM rats was due to the difficulty in growing rat embryonic stem cells (ESCs) in culture, a necessary first step in the most common methods of producing GM animals. Last year Matthew Evans wrote an article for the Pro-Test blog discussing how scientists at the University of Cambridge and the University of Southern California had developed a method for growing rat ESCs in culture, and how this achievement paved the way for the production of transgenic rats. Last week the same group of scientists announced that they had employed this method to produce GM rats whose p53 gene, a key tumor suppressor that is defective in several cancers, was deleted.

This is not the first time GM rats have been produced, as for the past few years scientists have been able to use zinc finger nucleases to knock-out rat genes. Zinc fingers, so called because one or more zinc ions stabilizes the finger like structure, are found in many proteins, allowing them to bind specifically to a structure within a cell, such as a particular DNA sequence. Scientists found that they could produce artificial zinc fingers that recognize particular genes, and then join a nuclease to that zinc finger so that it cuts out the target gene. This method, discussed in more detail in this excellent article by Elie Dolgin, allows scientists to knock-out genes in rat embryos. The downside of the zinc finger nuclease technique can only be used to knock-out genes, whereas the ESC method is more flexible – it can also be used to add extra copies of a gene, or to delete genes in specific tissues or stages of development.

It is now clear that the rat is joining the mouse at the forefront of the GM revolution in medicine, and that has to be great news for medical science and the patients that depend on it.

Paul Browne

1) Teslovich T.M. et al. "Biological, clinical and population relevance of 95 loci for blood lipids" Nature Volume 466, Pages 707-713 (2010) DOI:10.1038/nature09270

2) Musunuru K. et al. "From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus" Nature Volume 466, Pages 714–719 (2010) DOI:10.1038/nature09266

This morning the Home Office published the statistics on animal research for 2009 , and they make interesting reading. One change is that after a decade which has seen a steady rise in the number of animals used, and a sharp rise of 14% in 2008, the total number of procedures performed fell by 1% to just over 3.6 million. The rise in 2008 and subsequent fall in 2009 may represent a statistical blip, but it may also reflect the impact of the recession as projects were moved forward in anticipation of reduced funding in the future. With government funding of all scientific research in the UK facing cuts of up to 25% in the years ahead, it is likely that we will see further falls, since government funding through the research councils pays for about a third of projects involving animal research. After all, the increase we have observed over the past decade was due to a large extent to increases of about 50% in real terms in spending on biomedical research by the government, and we cannot now expect animal research to be immune from funding cuts. A lot will depend on whether spending by medical research charities and industry, which account for the remainder of the funding and also saw large increases in the past decade, can fill the gap left by government spending cuts.

One milestone passed this year was that for the first time over 50% of procedures involved the breeding or study of genetically modified animals (mainly rodents), which reflects a trend that we have noted before and reflects the growing importance they have to fields as diverse as cancer research and developing treatments for Duchenne Muscular Dystrophy.

One comment that stands out in the statement by Home Office Minister Lynne Featherstone is that the Government is“committed to ending the testing of household products on animals”, a frequent demand of animal rights groups. This would seem to be an easy promise to fulfill since, as our friends at Understanding Animal Research have pointed out, no such tests were performed in 2009, and very few, or none, in previous years of the past decade. The safety testing of household products on live animals has effectively already been ended by changes in the regulatory framework for chemicals, for example to the European REACH regulations and the recent development of alternatives that use cultured cells or tissues from dead animals and are sufficient for the evaluation of most cosmetics or household products. This is a perfect example of the 3Rs in action.

Overall this is a report that reflects both the profound changes that are happening in how and why animal research and toxicity testing is done, and the challenges facing British science as a whole in this time of austerity.

Turning to the activities of animal rights campaigners, we find that the Leicester Mercury has noted the tendency of a campaign by the National Anti-Vivisection Alliance* against a new laboratory at the University of Leicester to be economical with the truth. This will be no surprise to those of us who have been watching NAVA’s increasingly desperate attempts to stir up opposition to the new laboratory in Leicester, but will I’m sure be an eye-opener for Leicester citizens not yet familiar with degree to which some animal rights groups will misrepresent and distort the truth to advance their cause.

* NAVA is a new group, and is not to be confused with the more established National Anti-Vivisection Society

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.

Unlike previous microbicide gels that failed to offer significant protection against HIV infection this gel included the anti-retroviral drug tenofovir. Tenofovir was one of several anti-retroviral drugs discussed in an article on the role of non-human primate research in developing HIV prophylaxis by virologist Dr. Koen Van Rompay that was posted on the Speaking of Research blog 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 good news, Mel Broughton, former spokesperson for the animal rights group SPEAK that waged an often vicious (though occasionally bizarre) campaign against the new biosciences laboratory at Oxford, was jailed for 10 years last week for conspiracy to commit arson.

The unanimous verdict highlights once again how closely so-called "above ground" extremist campaings such as SPEAK and SHAC are connected to the criminality of those who operate under the banner of the ALF. Hopefully this verdict and sentence, along with the failure of Broughton's campaign to prevent the completion of the new laboratory, will help to dissuade other activists from resorting to such terror tactics.

* 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

Those of you who have been following the Pro-Test blog for a while will be aware that European Union (EU) is in the process of replacing Directive 86/609, the directive that covers the "laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes" with a new directive that better reflects the current state of the art of medical research and expected future developments, and does more to harmonize the regulations governing animal research in individual EU member states.

The proposed new directive on animal research was drafted by the European Commission in December 2008 and then sent to the European Parliament for its first reading. In common with the overwhelming majority of medical researchers in the EU we had grave concerns about some aspects of the proposed directive. Fortunately just before the June 2009 European elections, the EU parliament adopted a long list of amendments that it wanted incorporated, resolving many of the worries we had about the potential impact of the new directive on medical research in the EU. We were particularly pleased to see that the amendments protected the use of non-human primates in basic research, as these animals are of critical importance to fields such as neuroscience and virology.

This would usually have been sent to the European Council, a body that represents the governments of the individual EU member states, for them to give the proposal its first reading. However, to speed matters along, the Council initiated a trialogue procedure, in which representatives of the three parts of the EU legislature - the Commission, the Parliament and Council - hold private meetings to reach agreement on the text of the proposed directive.

The trialogue reached agreement on the text by December 2009, apart from a few details of committee procedure which needed to be clarified by legal experts, in the light of changes required by the Lisbon Treaty that entered into force on 1 December 2009. This agreement was marked by a formal letter from the European Parliament to the Council.

The clarification of the committee procedure points, followed by the legal checking of the text took until May, after which the Council agreed the text on 11 May and formally adopted it last week on 3 June.

We believe that the agreed directive strikes an excellent balance between the need to ensure that animals used in medical research are treated humanely, and the need to develop new treatments for terrible diseases and ensure that the EU remains at the forefront of medical science.

The next stage is that the draft directive will be sent back to the European Parliament for its second reading. Since the text is subject to a formal trialogue agreement this should be relatively swift and may involve as little as one vote in favour in the Agriculture Committee this summer and another at a plenary session of the full EU parliament probably in early autumn. At that point the Directive would be formally adopted into EU law.

There is certain to be some debate at each meeting and it is quite possible that the some EU parliamentary groups may put down amendments, but it is not thought likely that any will get the majority support they require. However, those experienced in the EU legislative process warn that one should always expect the unexpected. Some animal rights groups are continuing to lobby the parliament against the proposed text, so it would be a mistake to assume that its adoption is certain. That only happens when the ink is dry on the final document. We urge scientists to keep in touch with their MEP and to be ready to make the case for the well regulated use of animals in medical research.


Once the directive is adopted as EU legislation, all Member States must, within 2 years, pass (or amend existing) national legislation to make the provisions of the directive legally binding. It is possible that the UK parliament will need to amend the Animals (Scientific Procedures) Act 1986 that regulates animal research here, but it is more likely that any changes required will be achieved through changes to Home Office regulations, since nothing in the Animals (Scientific Procedures) Act 1986 appears to conflict with the new EU directive.

The adoption of the new directive by the EU council is excellent news, and represents one more step on the long and winding road towards an EU directive on animal research that we can all be proud of.