Tag Archives: animal testing

Nobel Prize 2015 – Protecting People against Parasites!

The 2015 Nobel Prize in Physiology or Medicine has been awarded to scientists whose research has led to therapies that have saved hundreds of millions of people around the world from parasitic diseases that can otherwise cause disability and death.

William C. Campbell and Satoshi Ōmura shared one half of the award “for their discoveries concerning a novel therapy against infections caused by roundworm parasites”, while Youyou Tu was awarded the other half “for her discoveries concerning a novel therapy against Malaria”.

Portraits of the winners of the Nobel medicine prize, shown on a screen at the ceremony in Stockholm. Photograph: Jonathan Nackstrand/AFP/Getty Images

Portraits of the winners of the Nobel medicine prize, shown on a screen at the ceremony in Stockholm. Photograph: Jonathan Nackstrand/AFP/Getty Images

In the press release announcing the award the Nobel Assembly at the Karolinska Institute highlighted the impact of the therapies that were developed thanks to the work done by these three scientists, Avermectin in the case of William C. Campbell and Satoshi Ōmura, and Artemisinin in the case of Youyou Tu.

The discoveries of Avermectin and Artemisinin have fundamentally changed the treatment of parasitic diseases. Today the Avermectin-derivative Ivermectin is used in all parts of the world that are plagued by parasitic diseases. Ivermectin is highly effective against a range of parasites, has limited side effects and is freely available across the globe. The importance of Ivermectin for improving the health and wellbeing of millions of individuals with River Blindness and Lymphatic Filariasis, primarily in the poorest regions of the world, is immeasurable. Treatment is so successful that these diseases are on the verge of eradication, which would be a major feat in the medical history of humankind. Malaria infects close to 200 million individuals yearly. Artemisinin is used in all Malaria-ridden parts of the world. When used in combination therapy, it is estimated to reduce mortality from Malaria by more than 20% overall and by more than 30% in children.

For Africa alone, this means that more than 100 000 lives are saved each year.

The discoveries of Avermectin and Artemisinin have revolutionized therapy for patients suffering from devastating parasitic diseases. Campbell, Ōmura and Tu have transformed the treatment of parasitic diseases. The global impact of their discoveries and the resulting benefit to mankind are immeasurable.

Animal research played a key part in the development of these therapies, as the Nobel Prize press release has pointed out.

Image: NobelPrize.org

Image: NobelPrize.org

In the case of Avermectin, after  Satoshi Ōmura had identified a series of bacterial cultures that produced a variety of antimicrobial agents, including the bacteria Streptomyces avermitilis which  showed promise against parasitic roundworm infection in mice, in 1979 William C. Campbell and colleagues identified a particular component produced by S. avermitilis called Avermectin B1a which had a broad efficiency against roundworm infections in a wide range of domesticated animal species, including cattle, sheep, dogs and chickens. Following this the team developed a modified form of Avermectin B1a known as Ivermectin, which was entered into clinical trials following positive tests in animal models of parasitic infection, and has since gone on to become a key treatment for parasitic infections – particularly the nematode worm infections that cause River Blindness and  Lymphatic Filariasis (the extreme manifestation of which is known as elephantitis) – and is on the World Health Organization’s list of Essential Medicines.

Image: NoberPrize.org

Image: NobelPrize.org

Ivermectin, and other members of the Avermectin family of therapies, are also widely used in veterinary practice, and their development and use is a good example of the One Health principle in action. You can learn more about the discovery of the Avermectins and Artemisinins in the advanced material Avermectin and Artemisinin – Revolutionary Therapies against Parasitic Diseases produced by the Nobel Assembly.

In 2011 we took a look at Professor Youyou Tu’s research that led to the development of Artemisinin therapy for malaria, and the key role played by mouse models of malaria infection,  in a post entitled “George is OK: Thank the men who stare down microscopes!”

While the news reports don’t state which drugs Cloony took to beat malaria, It is most likely that he was treated with artemisinin-based combination therapies (ACTs), which became available in the late 1990s and are now in widespread use.  If that is the case, he has benefited from mouse studies done in China the late 1960s and early 1970s when over 100 traditional herbal remedies were screened in a rodent model of malaria for anti-malarial activity (1). Eventually “Project 523” scored a hit when Professor Tu Youyou identified an extract of the plant qinghao, scientific name Artemisia annua, which had good anti-malarial activity, leading to the development of the artemisinin-based anti-malarials which have become the first-line treatment for malaria in the past decade.

We congratulate this years Nobel laureates in Physiology or Medicine, their research has improved the lives of hundreds of millions of people across the world over the past 3 decades, and will continue to do so. We hope that their success continues to inspire scientists around the world to rise to current and future public health challenges!

Speaking of Research

Truvada prevents HIV infection in high-risk individuals! A clinical success built on animal research

In the past two weeks we’ve learned of a major advance in ongoing efforts to halt the spread of  HIV, two separate clinical studies have reported that a daily regimen of a pill called Truvada as a pre-exposure prophylaxis (PrEP) is highly effective in preventing infection in high risk groups. This success is a result not just of the dedication of the clinicians who conducted these trials, but also of a series of pivotal studies conducted in non-human primates more than a decade ago that laid the scientific foundations for them.

In the first study of more than 600 high-risk individuals conducted at Kaiser Permanente in San Francisco, which was published in the journal Clinical Infectious Diseases, researchers found that Truvada – a combination of the anti-viral drugs tenofovir and emtricitabine – was 100% effective in preventing infection.  In the 2nd  study, called the PROUD study and published online this week in the Lancet, of more than 500 high-risk men undertaken in 13 sexual health clinics in England Truvada reduced infections by 86%.

Truvada prevents HIV transmission in high-risk individuals. Image: AFP / Kerry Sheridan

Truvada prevents HIV transmission in high-risk individuals. Image: AFP / Kerry Sheridan

These results have been greeted with enthusiasm in media reports, with headlines such as “Aids vanquished: A costly new pill promises to prevent HIV infection” , “A pill designed to prevent HIV is working even better than people thought” and  “Truvada Protected 100 Percent Of Study Participants From HIV: This is exciting!”. It’s worth noting that these are not the only trials to show the potential for Truvada to block HIV infection, earlier trials in Kenya, Uganda and Botswana also showed that it could substantially reduce infection rates, including in heterosexual couples where one partner was HIV positive and the other was not. There has been some concern that those taking Truvada would be less likely to take other safe sex measures – such as using condoms – but the results of the PROUD study showed no difference in acquisition of other sexually transmitted infections between those who started Truvada treatment immediately and those who delayed for 1 year, suggesting that they did not engage in riskier behavior as a consequence of taking Truvada.

Thanks to a multi-pronged approach to preventing HIV infection, combining barrier methods such as condoms,  Highly Active Antiretroviral Therapy (HAART) to lower viral load in infected individuals, and the use of antiviral medications to prevent mother-to-child transmission, the spread of HIV infection has slowed dramatically in many regions of the world, and pre-exposure prophylaxis with Truvada certainly has the potential to help reduce it further.

As we applaud the researchers who conducted these first real-world evaluations of Tenofovir in high-risk populations, it is also a good opportunity to remember the researchers whose work led us to this point. One of those pioneers is Dr. Koen Van Rompay, a virologist at the University of California at Davis who played a key role in the early development of Tenofovir and  its evaluation in pre- and post- exposure phophylaxis in macaque models of HIV infection. In 2009 Dr Van Rompay wrote an article for Speaking of Research explaining how important animal research was to the early development of such HIV prophylaxis regimes, and how important it continues to be as scientists develop ever better treatments, which we share again today:

Contributions of nonhuman primate studies to the use of HIV drugs to prevent infection – Koen van Rompay

Since the early days of the HIV pandemic, as soon as it was clear that an effective HIV vaccine would still be years away, there has been considerable interest in using anti-HIV drugs to reduce the risk of infection following exposure to HIV (so-called prophylaxis). Animal models of HIV infection, especially the rhesus macaque, have played a major role in developing and testing these treatments.

The development of HIV drugs to treat HIV-infected persons has shown that many compounds that are effective in vitro (i.e., in tissue culture assays) fail to hold their promise when tested in humans, because of unfavorable pharmacokinetics, toxicity or insufficient antiviral efficacy. The same principles apply to the development of drugs to prevent HIV infection. The outcome of drug administration is determined by many complex interactions in vivo between the virus, the antiviral drug(s) and the host; with current knowledge, these interactions cannot be mimicked and predicted sufficiently by in vitro studies or computer models.

Testing different compounds in human clinical trials is logistically difficult, time-consuming and expensive, so only a very limited number of candidates can be explored in a given time. Fortunately, the development of antiviral strategies can be accelerated by efficient and predictive animal models capable of screening and selecting the most promising compounds. No animal model is perfect and each model has its limitations, but the simian immunodeficiency virus (SIV) of macaques is currently considered the best animal model for HIV infection because of the many similarities of the host, the virus and the disease. Non-human primates are phylogenetically the closest to humans, and have similar immunology and physiology (including drug metabolism, placenta formation, fetal and infant development). In addition, SIV, a virus closely related to HIV-1, can infect macaques and causes a disease that resembles HIV infection and AIDS in humans, and the same markers are used to monitor the disease course. For these reasons, SIV infection of macaques has become an important animal model to test antiviral drugs to prevent or treat infection.

Studies in rhesus macaques first indicated that Tenofovir could block HIV infection. Photo: Understanding Animal Research

Studies in rhesus macaques first indicated that Tenofovir could block HIV infection. Photo: Understanding Animal Research

Different nonhuman primate models have been developed based on the selection of the macaque species, the particular SIV strain and the inoculation route (e.g. IV injection, vaginal exposure) used (reviewed in (33)). These models have been improved and refined during the past two decades. For example, SIV-HIV chimeric viruses have been engineered to contain portions of HIV-1, such as the enzyme reverse transcriptase (“RT-SHIV”) that the virus requires in order to multiply or the envelope protein (“env-SHIV”) that the virus needs if it is to escape from a cell and infect other cells, to allow these models to also test drugs that are specific for HIV-1 reverse transcriptase or envelope (28, 35).

Many studies in non-human primates have investigated whether the administration of anti-HIV drugs prior to or just after exposure to virus can prevent infection. The earliest studies indicated that drugs such as the reverse transcriptase inhibitor zidovudine (AZT), the first approved drug treatment for HIV, were not very effective in preventing infection, but a likely reason for this was the combination of a high-dose viral inoculums used, the direct intravenous route of virus inoculation, and the relative weak potency of drugs at that time (2, 4, 13, 19, 20, 36). The proof-of-concept that HIV drugs can prevent infection was demonstrated in 1992 when a 6-weeks zidovudine regimen, started 2 hours before an intravenous low-dose virus inoculation that more accurately represented HIV infection in humans, protected infant macaques against infection (29). These results were predictive of a subsequent clinical trial (Pediatric AIDS Clinical Trials Group Protocol 076), which demonstrated that zidovudine administration to HIV-infected pregnant women beginning at 14 to 34 weeks of gestation, and continuing to their newborns during the first 6 weeks of life reduced the rate of viral transmission by two-thirds (10).

Since then, a growing number of studies have been performed in macaques to identify more effective and simpler prophylactic drug regimens. These studies generally used lower virus doses, sometimes combined with a mucosal route of virus inoculation that mimics vaginal or anal exposure responsible for the majority of human HIV infections. These studies demonstrated that administration of some newer anti-HIV drugs, including the reverse transcriptase inhibitors adefovir (PMEA), tenofovir (PMPA), and emtricitabine (FTC) that prevent the virus from multiplying in the infected cell, and the CCR5 inhibitor CMPD167 that stops the virus from binding the CCR5 receptor on the cell surface and entering a cell in the first place, starting prior to, or at the time of virus inoculation, was able to prevent infection, though with varying success rates (3, 4, 16, 24, 25, 31, 34, 35). Only very few compounds such as the reverse transcriptase inhibitors tenofovir, BEA-005 and GW420867, and the CCR5 inhibitor CMPD167, were able to reduce infection rates when treatment was started after virus inoculation. For those drugs that were successful in post-exposure prophylaxis studies, a combination of the timing and duration of drug administration was found to determine the success rate, because a delay in the start, a shorter duration, or interruption of the treatment regimen all reduced the prophylactic efficacy (5, 11, 21, 22, 26, 27, 31) , information that has guided the design of subsequent clinical trials.

While some of the compounds such as GW420867 that showed prophylactic efficacy in the macaque model are no longer in clinical development (e.g., due to toxicity or pharmacokinetic problems discovered later in pre-clinical testing), the very promising results achieved with tenofovir have sparked further studies aimed at simplifying the prophylactic regimen. Several studies in infant and adult macaques have demonstrated that short or intermittent regimens of tenofovir (with or without coadministration of emtricitabine) consisting of one dose before and one dose after each virus inoculation were highly effective in reducing SIV infection rates (15, 30, 32).

The demonstration at the beginning of the 1990’s that anti-HIV drugs can prevent infection in macaques has provided the rationale to administer these compounds to humans to reduce the likelihood of infection in several clinical settings. Antiviral drugs are now recommended, usually as a combination of several drugs, to reduce the risk of HIV infection after occupational exposure (e.g., needle-stick accidents of health care workers) and non-occupational exposure (e.g. sex or injection-drug use) (6, 7). As mentioned previously, drug regimens containing zidovudine and more recently also more potent drugs such as nevirapine have proven to be highly effective in reducing the rate of mother-to-infant transmission of HIV, including in developing countries (10, 14, 17), and save many thousands of lives every year . Because the short nevirapine regimen that is given to pregnant HIV-infected women at the onset of labor frequently induces drug resistance mutations in the mother that may compromise future treatment (12), tenofovir’s high prophylactic success in the infant macaque model has sparked clinical trials in which a short tenofovir-containing regimen was added to existing perinatal drug regimens to reduce the occurrence of resistance mutations and/or further lower the transmission rate (8, 9, 18, 30, 32).

Scanning electron micrograph of HIV-1, colored green, budding from a cultured lymphocyte. Photo: C. Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus

Scanning electron micrograph of HIV-1, colored green, budding from a cultured lymphocyte. Photo: C. Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus

Because an efficacious HIV vaccine has so far not been identified, the concept of using pre-exposure prophylaxis also as a possible HIV prevention strategy in adults has gained rapid momentum in recent years. The promising prophylactic data of tenofovir (with or without emtricitabine) in the macaque model (23, 32, 35, 37) combined with the favorable pharmacokinetics, safety profile, drug resistance pattern and therapeutic efficacy of these drugs in HIV-infected people, have pushed these compounds into front-runner position in ongoing clinical trials that investigate whether uninfected adults who engage in high-risk behavior will have a lower infection rate by taking a once daily tablet of tenofovir or tenofovir plus emtricitabine. The results of these ongoing trials are highly anticipated. An overview of the design, status and challenges of these trials which are currently underway at several international sites and target different high-risk populations can be found on the website of the AIDS Vaccine Advicacy Coalition (1, 23).

In conclusion, nonhuman primate models of HIV infection have played an important role in guiding the development of pre- and post-exposure prophylaxis strategies. Ongoing comparison of results obtained in these models with those observed in human studies will allow further validation and refinement of these animal models so they can continue to provide a solid foundation to advance our scientific knowledge and to guide clinical trials.

Koen van Rompay DVM Ph.D. is a research virologist at the California National Primate Research Center at UC Davis.

Cited literature
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14. Gaillard, P., M.-G. Fowler, F. Dabis, H. Coovadia, C. van der Horst, K. Van Rompay, A. Ruff, T. Taha, T. Thomas, I. de Vicenzi, M.-L. Newell, and for the Ghent IAS Working Group on HIV in Women and Children. 2004. Use of antiretroviral drugs to prevent HIV-1 transmission through breastfeeding: from animal studies to randomized clinical trials. J. Acquired Immune Defic. Syndr. 35:178-187.
15. Garcia-Lerma, J. G., R. A. Otten, S. H. Qari, E. Jackson, M. E. Cong, S. Masciotra, W. Luo, C. Kim, D. R. Adams, M. Monsour, J. Lipscomb, J. A. Johnson, D. Delinsky, R. F. Schinazi, R. Janssen, T. M. Folks, and W. Heneine. 2008. Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir. PLoS Med. 5:e28.
16. Grob, P. M., Y. Cao, E. Muchmore, D. D. Ho, S. Norris, J. W. Pav, C.-K. Shih, and J. Adams. 1997. Prophylaxis against HIV-1 infection in chimpanzees by nevirapine, a nonnucleoside inhibitor of reverse transcriptase. Nature Med. 3:665-670.
17. Guay, L. A., P. Musoke, T. Fleming, D. Bagenda, M. Allen, C. Nakabiito, J. Sherman, P. Bakaki, C. Ducar, M. Deseyve, L. Emel, M. Mirochnick, M. G. Fowler, L. Mofenson, P. Miotti, K. Dransfield, D. Bray, F. Mmiro, and J. B. Jackson. 1999. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomized trial. Lancet 354:795-802.
18. Hirt, D., S. Urien, D. K. Ekouevi, E. Rey, E. Arrive, S. Blanche, C. Amani-Bosse, E. Nerrienet, G. Gray, M. Kone, S. K. Leang, J. McIntyre, F. Dabis, and J. M. Treluyer. 2009. Population pharmacokinetics of tenofovir in HIV-1-infected pregnant women and their neonates (ANRS 12109). Clin. Pharmacol. Ther. 85:182-9.
19. Lundgren, B., D. Bottiger, E. Ljungdahl-Ståhle, E. Norrby, L. Ståhle, B. Wahren, and B. Öberg. 1991. Antiviral effects of 3′-fluorothymidine and 3′-azidothymidine in cynomolgus monkeys infected with simian immunodeficiency virus. J. Acquir. Immune Defic. Syndr. 4:489-498.
20. McClure, H. M., D. C. Anderson, A. A. Ansari, P. N. Fultz, S. A. Klumpp, and R. F. Schinazi. 1990. Nonhuman primate models for evaluation of AIDS therapy. Ann. N. Y. Acad. Sci. 616:287-298.
21. Mori, K., Y. Yasumoti, S. Sawada, F. Villinger, K. Sugama, B. Rosenwirth, J. L. Heeney, K. Überla, S. Yamazaki, A. A. Ansari, and H. Rübsammen-Waigmann. 2000. Suppression of acute viremia by short-term postexposure prophylaxis of simian/human immunodeficiency virus SHIV-RT-infected monkeys with a novel reverse transcriptase inhibitor (GW420867) allows for development of potent antiviral immune responses resulting in efficient containment of infection. J. Virol. 74:5747-5753.
22. Otten, R. A., D. K. Smith, D. R. Adams, J. K. Pullium, E. Jackson, C. N. Kim, H. Jaffe, R. Janssen, S. Butera, and T. M. Folks. 2000. Efficacy of postexposure prophylaxis after intravaginal exposure of pig-tailed macaques to a human-derived retrovirus (human immunodeficiency virus type 2). J Virol 74:9771-5.
23. PrEP Watch, http://www.prepwatch.org/
24. Subbarao, S., R. A. Otten, A. Ramos, C. Kim, E. Jackson, M. Monsour, D. R. Adams, S. Bashirian, J. Johnson, V. Soriano, A. Rendon, M. G. Hudgens, S. Butera, R. Janssen, L. Paxton, A. E. Greenberg, and T. M. Folks. 2006. Chemoprophylaxis with Tenofovir Disoproxil Fumarate Provided Partial Protection against Infection with Simian Human Immunodeficiency Virus in Macaques Given Multiple Virus Challenges. J. Infect. Dis. 194:904-11.
25. Tsai, C.-C., K. E. Follis, A. Sabo, R. F. Grant, C. Bartz, R. E. Nolte, R. E. Benveniste, and N. Bischofberger. 1994. Preexposure prophylaxis with 9-(-2-phosphonylmethoxyethyl)adenine against simian immunodeficiency virus infection in macaques. J. Infect. Dis. 169:260-266.
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28. Uberla, K., C. Stahl-Hennig, D. Böttiger, K. Mätz-Rensing, F. J. Kaup, J. Li, W. A. Haseltine, B. Fleckenstein, G. Hunsmann, B. Öberg, and J. Sodroski. 1995. Animal model for the therapy of acquired immunodefiency syndrome with reverse transcriptase inhibitors. Proc. Natl. Acad. Sci. U.S.A. 92:8210-8214.
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30. Van Rompay, K. K. A., C. J. Berardi, N. L. Aguirre, N. Bischofberger, P. S. Lietman, N. C. Pedersen, and M. L. Marthas. 1998. Two doses of PMPA protect newborn macaques against oral simian immunodeficiency virus infection. AIDS 12:F79-F83.
31. Van Rompay, K. K. A., M. L. Marthas, J. D. Lifson, C. J. Berardi, G. M. Vasquez, E. Agatep, Z. A. Dehqanzada, K. C. Cundy, N. Bischofberger, and N. C. Pedersen. 1998. Administration of 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) for prevention of perinatal simian immunodeficiency virus infection in rhesus macaques. AIDS Res. Hum. Retroviruses 14:761-773.
32. Van Rompay, K. K. A., M. B. McChesney, N. L. Aguirre, K. A. Schmidt, N. Bischofberger, and M. L. Marthas. 2001. Two low doses of tenofovir protect newborn macaques against oral simian immunodeficiency virus infection. J. Infect. Dis. 184:429-438.
33. Van Rompay, K. K. A. 2005. Antiretroviral drug studies in non-human primates: a valid animal model for innovative drug efficacy and pathogenesis studies. AIDS Reviews 7:67-83.
34. Van Rompay, K. K. A., B. P. Kearney, J. J. Sexton, R. Colón, J. R. Lawson, E. J. Blackwood, W. A. Lee, N. Bischofberger, and M. L. Marthas. 2006. Evaluation of oral tenofovir disoproxyl fumarate and topical tenofovir GS-7340 to protect infant macaques against repeated oral challenges with virulent simian immunodeficiency virus. J. Acquir. Immune Defic. Syndr. 43:6-14.
35. Veazey, R. S., M. S. Springer, P. A. Marx, J. Dufour, P. J. Klasse, and J. P. Moore. 2005. Protection of macaques from vaginal SHIV challenge by an orally delivered CCR5 inhibitor. Nat Med.
36. Wyand, M. S. 1992. The use of SIV-infected rhesus monkeys for the preclinical evaluation of AIDS drugs and vaccines. AIDS Res. Hum. Retrovir. 8:349-356.
37. García-Lerma J. G., Otten R. A., Qari S. H., Jackson E., Cong M. E., Masciotra S., Luo W., Kim C., Adams D. R., Monsour M., Lipscomb J., Johnson J. A., Delinsky D., Schinazi R. F., Janssen R , Folks T. M., Heneine W. Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir. PLoS Med. 2008 Feb;5(2):e28


Israel provides animal research statistics for 2014

The 2014 statistics from the Israeli National Council for Animal Experimentation show a 13 percent increase in animals used, reports Haaretz, an Israeli newspaper.

The 340,330 animals used in experimentation in 2014 represent the highest animal use since 2007, the peak of animal experimentation in Israel. Rodents comprised the majority (84 percent) of the animals used for experiments, birds and fish came next with around 7% each, while larger mammals accounted for only 1.3 percent of the total. The number of mice used, 236,000, represents a 12 percent increase over the 2013 amount.

Animals used in research in Israel 2010-14

For the second year in a row, no dogs or cats were used as experimental subjects. More monkeys were used for experimentation than in previous years; however, the National Council for Animal Experimentation report notes that Israel, with a rehabilitation rate of 89 percent, ranks among the countries with the highest reintegration rates for monkeys.

Dogs cats monkeys used in Israel 2010-2014

Seven percent of the animals were fish, which represents a three-fold increase over the previous year. The report by the National Council for Animal Experimentation attributes this increase to a concerted effort to use the lowest animal on the “developmental scale” that is scientifically appropriate.

On the five point pain scale, 12 percent of experimental animals were exposed to the highest amount of pain and 19 percent were ranked in the lowest pain category. Strict supervision of the animals by veterinarians and unannounced laboratory inspections prevent unnecessary pain for the animals, The Jerusalem Post reports.

Medical and scientific research were the main uses for the animals, accounting for 46 and 45 percent, respectively. Testing new products and materials used eight percent of the animals, and one percent was used for teaching.

Transparent reports of animal use contribute to public education about animal research. Speaking of Research continues to report on these statistical reports as they come out, most recently the 2014 statistics for the United States and Ireland and the 2012 Canadian report.

Alyssa Ward

USDA publishes 2014 Animal Research Statistics

Congratulations to the USDA/APHIS for getting ahead of the curve and making the US the first country to publish its 2014 animal research statistics. Overall, the number of animals (covered by the Animal Welfare Act) used in research fell 6.4% from 891,161 (2013) to 834,453 (2014).

These statistics do not include all animals as most mice, rats, and fish are not covered by the Animal Welfare Act – though they are still covered by other regulations that protect animal welfare. We also have not included the 166,274 animals which were kept in research facilities in 2014 but were not involved in any research studies.

Types of Animals used in research and testing 2014Statistics from previous years show that most of the “All other animals” species are rodents (but not mice or rats). 53% of research is on guinea pigs, hamsters and rabbits, while 10% is on dogs or cats and 7% on non-human primates. In the UK, where mice, rats, fish and birds are counted in the annual statistics, over 97% of research is on rodents, birds and fish. Across the EU, which measures animal use slightly differently, 93% of research is on species not counted under the Animal Welfare Act. We would expect similar patterns to be true in the US – although there are no statistics to confirm this.

Changes in number of animals used in research from 2013 to 2014 - Click to Enlarge

Changes in number of animals used in research from 2013 to 2014 – Click to Enlarge

If we look at the changes between the 2013 and 2014 statistics we can see a drop in the number of animals of most species , with only the “all other animals” category showing a rise. This is the second year in which the number of many species has fallen. For example, the number of rabbits used in 2014 fell 11.4% from 2013, following a 9.2% fall from 2012.

Most notably the number of non-human primates has fallen by 9.9%, the number of dogs fell 12.4% and the number of cats fell by 13%. This has shown these species taking up a smaller proportion of the research animals used, as can be seen below:

Trend in number of animals used in research 1973 - 2014 - Click to Enlarge

Trend in number of animals used in research 1973 – 2014 – Click to Enlarge

Clearly there has been a downward trend in the number of animals used since the early 1990s with a 61% drop in numbers between 1992 and 2014. It is also likely that, similar to the UK, a move towards using more genetically altered mice and fish has reduced the numbers of other AWA-covered animals used.

Rises and falls in the number of animals used reflects many factors including the level of biomedical activity in a country, trending areas of research, changes to legislations at home and abroad, outsourcing research to and from other countries, and new technologies (which may either replace animal studies or create reasons for new animal experiments).

It is important to note that the number of animals cannot be tallied across years to get an accurate measure of total number of animals. This is because animals in longitudinal studies are counted each year. Thus, if the same 10 animals are in a research facility for 10 years, they would appear in the stats of each year – adding these numbers would incorrectly create the illusion of 100 animals being used.

Speaking of Research welcomes the open publication of these animal research statistics as offering the public a clear idea of what animal research goes on in their country.

Clinical trial success for Cystic Fibrosis gene therapy: built on animal research

This morning the Cystic Fibrosis Gene Therapy Consortium (GTC) announced the results of clinical trial in 140 patients with cystic fibrosis, which demonstrate the potential for gene therapy to slow – and potentially halt – the decline of lung function in people with the disorder. It is a success that is built on 25 years of research, in which studies in animals have played a crucial role.

Cystic fibrosis is one of the most commonly inherited diseases, affecting about one in every four thousand children born in the USA, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene produces a channel that allows the transport of chloride ions across membranes in the body, and the many mutations identified in cystic fibrosis sufferers either reduce the activity of the channel or eliminate it entirely. This defect in chloride ion transport leads to defects in several major organs including the lungs, digestive system, pancreas, and liver. While the severity of the disease and the number of organs affected varies considerably, cystic fibrosis patients often ultimately require lung transplant s, and too many still die early in their 20’s and 30’s as the disease progresses.

In a paper published in Lancet Respiratory Medicine today (1), the GTG members led by Professor Eric Alton of Imperial College London compared monthly delivery to the airway of a non-viral plasmid vector containing the CFTR gene in the liposome complex pGM160/GL67A using a nebuliser with a placebo group who received saline solution via the nebuliser. They reported stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group after a year. This is the first time that gene therapy has been shown to safely stabilise the disease, and while the difference between the treated and control group was modest, and the therapy is not yet ready to go into clinical use, it provides a sound bases for further development and improvement.


The Chief Executive of the Cystic Fibrosis Trust, which is one of the main funders of the GTC, has welcomed the results, saying:

Further clinical studies are needed before we can say that gene therapy is a viable clinical treatment. But this is an encouraging development which demonstrates proof of concept.

“We continue to support the GTC’s ground-breaking work as well as research in other areas of transformational activity as part of our mission to fight for a life unlimited by cystic fibrosis.”

So how did animal research pave the way for this trial?

Following the identification of the CFTR gene in 1989 scientists sought to create animal models of cystic fibrosis with which to study the disease, and since the early 1990’s more than a dozen mouse models of cystic fibrosis have been created. In some of these the CFTR gene has been “knocked out”, in other words completely removed, but in others the mutations found in human cystic fibrosis that result in a defective channel have been introduced. These mouse models show many of the defects seen in human cystic fibrosis patients and over the past few years have yielded important new information about cystic fibrosis, and in 1993 Professor Alton and colleagues demonstrated that it is possible to deliver a working copy of the CFTR gene using liposomes to the lungs of CFTR knockout mice and correct some of the deficiencies observed.

To get a working copy of the CFTR gene to the lungs of cystic fibrosis patients Professor Alton and colleagues needed three things:

• A DNA vector containing the working CFTR gene that is safe and  can express sufficient amounts of the CFTR channel protein in the lungs to correct the disease

• A lipid-like carrier that can form a fatty sphere around the DNA vector to so that it can cross the lipid membrane of cells in the lung, as “naked” DNA will not do this efficiently.

• A nebuliser device that produces an aerosol of the gene transfer agent so that it can be inhaled into the lungs of the patient.

Several early attempts to use gene therapy using viral vectors to deliver the working copy of the CFTR gene to patients failed because the immune response rapidly neutralised the adenoviral vector (see this post for more information on challenges using adenoviral vectors), and while attempts to use non-viral vectors were more promising, it was found that they caused a mild inflammation in most patients, which would make then unsuitable for long term use. As reported in a paper published in 2008 the GTC members developed and assessed in mice a series of non-viral DNA vectors, repeatedly modifying them and testing their ability to both drive CFTR gene expression in the lungs and avoid inducing inflammation. They finally hit on a vector – named pGM169 – which fulfilled both key criteria.

Earlier the consortium had undertaken a study to determine which carrier molecule to use in their non-viral gene transfer agent (GTA). To do this they assessed 3 GTA’s, each consisting of a lipid like molecule that could form a sphere around the non-viral DNA vector; either the 25 kDa-branched polyethyleneimine (PEI), the cationic liposome GL67A, or as a compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. Because there are significant differences in airway physiology between mouse and human they carried out this study in sheep, whose lung physiology more closely matches that of humans. The study identified the cationic liposome GL67A as the most promising candidate, resulting in robust expression of the CFTR transgene in the sheep lungs.

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

It now remained to bring the DNA vector and carrier together. In a 2013 publication the consortium reported that repeated aerosol doses of pGM169/GL67A to sheep over a 32 week period were safe and induced expression of the CFTR transgene in the sheep lungs, although the level of expression varied between individuals (this variation was also observed in human CF patients in the clinical trial reported today). A final study, this time in mice, assessed the suitability of the Trudell AeroEclipse II nebuliser as a device to create stable pGM169/GL67A aerosols, finding that it did so in a reproducible fashion. When aerosolized to the mouse lung, the new pGM169/GL67A formulation was capable of directing persistent CFTR transgene expression for at least 2 months, with minimal inflammation. These studies provided the evidence to support the gene delivery system and dosage strategy used in the clinical trial reported today.

The trial results announced today are an important accomplishment, but they mark a beginning rather than the end for Cystic Fibrosis gene therapy. It will be necessary to improve the efficiency of the therapy before it can enter widespread clinical use. Animal research will certainly play an important part in this work, notably the observation that the efficiency of CFTR gene delivery using this strategy was varied between individuals in both sheep and humans indicates that sheep are a good model in which to assess changes to improve the consistency and effectiveness of the gene therapy.

If you would like to know more about this cystic fibrosis gene therapy clinical trial you can watch two videos recorded at a meeting for cystic fibrosis patients at ICL on the  Cystic Fibrosis Trust website.

Paul Browne

1) Alton E.W.F.W. et al. “Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial” Lancet Respiratory Medicine Published online July 3, 2015

European Commission rejects Stop Vivisection Initiative

Today the European Commission rejected the Stop Vivisection Initiative that sought to repeal European Directive 2010/63/EU on the protection of animals used for scientific purposes and ban animal research in the EU.

Today, there are effective treatments for many infectious diseases, some forms of cancer, and several chronic diseases such as diabetes. These advancements would have been impossible without the insights gained in animal studies.
However, the Commission does not share the view that scientific principles invalidate the ‘animal model’. Indeed, despite differences with humans, animal models have been the key scientific drivers to develop almost all existing effective and safe medical treatments and prevention measures for human and animal diseases
The Commission therefore does not intend to submit a proposal to repeal Directive 2010/63/EU and is not intending to propose the adoption of a new legislative framework.

Read the full EU report here.

Dr Paul Browne, Research Editor at Speaking of Research, said:

We welcome the decision by the European Commission to reject the Stop Vivisection Initiative. EU Directive 2010/63 which governs animal experiments has been a step forward for both animal welfare and better science. They put the 3Rs – Replacement, Refinement and Reduction of animals in research – at the heart of the rules governing animal experiments.

Animal research continues to play a key part in medical advances. Only last week we learned about a new lung cancer therapy that performed very well in clinical trials, allowing patients with the disease to live longer; this treatment was only possible thanks to studies in transgenic mice. “

The Commission’s decision is not, however, unexpected. Directive 2010/63/EU was adopted by the EU Council and Parliament in September 2010 after more than 5 years of discussion and debate, including consultation exercises in which scientists, patient organizations, animal welfare experts, animal rights organizations and members of the public were given the opportunity to submit evidence. At a time when the EU is facing some of the greatest political and economic challenges of its history it was always very unlikely that the EU commission would repeal Directive 2010/63/EU and start the negotiation process again from scratch.


If the organizers and supporters of the Stop Vivisection Initiative were going to have any chance of persuading the Commission to repeal directive 2010/63/EU, they needed to make a very strong case to the MEPs who gathered to hear what they had to say at the European Parliament session held on Monday 11 May 2015.

They didn’t. The hearing was something of a flop, with reports noting that the majority of MEPs present were unconvinced by the arguments put forward by the proponents of the Stop Vivisection Initiative. It’s not difficult to see why this was the case. The Stop Vivisection Organizers and their witnesses failed to put forward any significant new evidence that had not been examined back when the Directive 2010/63/EU was originally negotiated, and at one point in the hearing descended into outright conspiracy theory thinking.

By contrast supporters of Directive 2010/63/EU made a stronger case, especially Nobel laureate Professor Francoise Barré – Sinoussi, who put forward a very strong case for the value of animal research in advancing medicine.

While this was happening scientists and supporters of medical progress in the EU were not taking any chances, and let the European Commission know in no uncertain terms how important animal research is to medical science. More than 170 organizations (Speaking of Research among them) representing scientists, major funders of medical research  and many millions of patients across the EU have signed up to a statement in support of Directive 2010/63/EU and sixteen European Nobel laureates published an open letter in UK and German newspapers to rebut the Stop Vivisection campaign. Several excellent letters on the importance of animal research were published in the national press, including a letter in the Times by Steve Ford, Chief executive of Parkinson’s UK, as well as articles such as that written by Oxford University Duchenne muscular dystrophy researcher Professor Kay Davies. In addition research funders have added information explaining their position on animal research to their websites, for example the Wellcome Trust, one of the world’s top medical research charities, have published a briefing on “Why we support research involving animals”, and a Q&A on European Directive 2010/63/EU.

We congratulate the European Commission on this good decision for science and patients in Europe, and the EU scientific community for speaking up for science with one voice.

Speaking of Research

Lung cancer immunotherapy, from PD-1 knockout mice to clinical trials

This morning many news outlets, including the BBC, covered a very promising development in lung cancer therapy; the successful clinical trial of the cancer immunotherapy Nivolumab in 582 patients with advanced lung cancer. While the extension of survival was modest in most patients, it is to be remembered that these were patients with advanced lung cancer, which is notoriously difficult to treat, so to see the survival time doubling in some patients was quite dramatic. Future trials will examine whether greater benefits are seen when Nivolumab is given earlier in the course of the disease.

Dr Alan Worsley, Cancer Research UK’s senior science information officer, told the BBC that harnessing the immune system would be an “essential part” of cancer treatment, and adding:

This trial shows that blocking lung cancer’s ability to hide from immune cells may be better than current chemotherapy treatments. “Advances like these are giving real hope for lung cancer patients, who have until now had very few options.”

Nivolumab works by blocking the activation of the PD-1 receptor protein found on the surface of many of the immune cells that infiltrate tumours. Another protein named PD-L1 binds to PD-1 and initiates a regulatory pathway that leads to the immune response being dampened down. Usually this is a good thing as it maintains immune tolerance to self-antigens and prevents auto-immune damage to healthy tissue, but unfortunately many solid tumour cells, such as lung cancer cells, also secrete PD-L1, and by activating PD-1 can evade destruction by the immune system. By blocking PD-1 Nivolumab turns off this protective mechanism and allows the immune cells to detect and destroy the tumour cells.

X-ray of a lung cancer patient. Image credit:

X-ray of a lung cancer patient. Image credit: “LungCACXR” by James Heilman, MD – Own work.

So how was this discovered? This is where the knockout mice come in. Scientists had observed in the 1990’s that PD-1 was highly expressed on the surface of circulating T- and B- immune cells in mice, but didn’t know what role PD-1 played, suspecting that it may be involved in increasing the magnitude of the immune response. To examine the role of PD-1 researchers at Kyoto University in Japan led by Professor Tasuku Honjo created a knock-out mouse line where the PD-1 gene was absent, and observed that this lead to some immune responses being augmented. In a paper published in 1998 they reported than rather than being an activator of the immune response PD-1 was actually involved in dampening down the immune response (1).

Subsequent studies in a range of PD-1 knockout mouse strains over the next decade explored the role of PD-1 in regulating the immune system, and also demonstrated that its ligand, PD-L1, could block immune-mediated tissue damage (2).  At the same time as these studies were taking place other research was demonstrating that PD-L1 was produced at high levels by tumour cells, first in   renal cell carcinoma in 2004 (3), but later in many other solid tumours including in lung cancer (4), and that this expression was associated with a decrease in the immune response to the tumour and a poorer prognosis.

This raised an obvious question: would blocking PD-1 improve the immune response against these tumours?

Work was already underway to find out. A paper published in 2007 by scientists from Nara Medical University in Japan demonstrated that blocking PD-L1 binding to PD-1 with monoclonal antibodies enhanced the immune response against established tumours in a mouse model of pancreatic cancer and acted synergistically with chemotherapy to clear the tumours without obvious toxicity (5). Subsequent studies with other monoclonal antibodies in a range of mouse and in vitro models of cancer showed similar results, including the humanized monoclonal antibody MDX-1106, now called Nivolumab, which was obtained by immunizing mice which had been genetically modified to produce human antibodies with human PD-1 (6).

Laboratory Mice are the most common species used in research

Cancer Immunotherapy – adding another accomplishment to an already impressive CV!

MDX-1106/Nivolumab showed promising results in a phase 1 trial against metastatic melanoma, colorectal cancer, castrate-resistant prostate cancer, non-small-cell lung cancer, and renal cell carcinoma, and following larger clinical trials (7) it was approved by the FDA for the treatment of melanoma that cannot be removed by surgery or is metastatic and no longer responding to other drugs, and more recently for metastatic squamous non-small cell lung cancer.

The story of the development of anti-PD-1 cancer immunotherapy is an illustration of how basic or fundamental biological research in animals informs medical science, and drives the discovery of new therapies. As cancer immunotherapy begins to transform the treatment of many previously untreatable cancers, it is well worth remembering that this revolution has its origin in the hard work of countless scientists working around the world, many of whom could only have guessed at the time where their efforts would eventually lead.

Breaking news, 1 June 2015: In another exciting report from the American Society of Clinical Oncology meeting in Chicago, researchers have reported that in a clinical trial of 945 patients with advanced metastatic melanoma a combination of Nivolumab with  Ipilimumab (another cancer immunotherapy that works through a different mechanism) stopped cancer advancing for nearly a year in 58% of cases, with the cancer still stopped in its tracks in many patients when the study period had ended. This is substantially greater effect than is seen with existing therapies, including Ipilimumab when administered alone, and shows how powerful cancer immunotherapies may be when two or more are combined.

Paul Browne


  1. Nishimura H1, Minato N, Nakano T, Honjo T. “Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses.” Int Immunol. 1998 Oct;10(10):1563-72. PubMed: 9796923
  2. Grabie N, Gotsman I, DaCosta R, Pang H, Stavrakis G, Butte MJ, Keir ME, Freeman GJ, Sharpe AH, Lichtman AH. “Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart.” Circulation. 2007 Oct 30;116(18):2062-71. PubMed 17938288
  3. Thompson RH1, Gillett MD, Cheville JC, Lohse CM, Dong H, Webster WS, Krejci KG, Lobo JR, Sengupta S, Chen L, Zincke H, Blute ML, Strome SE, Leibovich BC, Kwon ED. “Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target.” Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17174-9. PubMed:15569934
  4. Zhang Y1, Huang S, Gong D, Qin Y, Shen Q. “Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer.” Cell Mol Immunol. 2010 Sep;7(5):389-95. doi: 10.1038/cmi.2010.28. PubMed: 20514052
  5. Nomi T1, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H, Nakamura S, Enomoto K, Yagita H, Azuma M, Nakajima Y. “Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer.” Clin Cancer Res. 2007 Apr 1;13(7):2151-7. PubMed:17404099
  6. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman WH, Stankevich E, Pons A, Salay TM, McMiller TL, Gilson MM, Wang C, Selby M, Taube JM, Anders R, Chen L, Korman AJ, Pardoll DM, Lowy I, Topalian SL. “Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.” J Clin Oncol. 2010 Jul 1;28(19):3167-75. doi:10.1200/JCO.2009.26.7609. PubMed: 20516446
  7. Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, Brahmer JR, Lawrence DP, Atkins MB, Powderly JD, Leming PD, Lipson EJ, Puzanov I, Smith DC, Taube JM, Wigginton JM, Kollia GD, Gupta A, Pardoll DM, Sosman JA, Hodi FS. “Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab.” J Clin Oncol. 2014 Apr 1;32(10):1020-30. doi: 10.1200/JCO.2013.53.0105. PubMed:24590637