Huntington’s disease. Dr. Steven Finkbeiner.

In the June 10, 2011 edition of Cell, there is a Leading Edge Preview (short review) and a research Article on a surprising new potential therapeutic strategy for neurodegenerative disease. The Preview is by Peter H Reinhart (Proteostasis Therapeutics, Cambridge MA) and Jeffery W Kelly (Skaggs Institute and Scripps Research Institute, La Jolla CA), and the the Article (Zwilling et al.) is by Paul J Muchowski (Gladstone Institute of Neurological Disease, University of California at San Francisco) and his colleagues. In addition to Dr. Muchowski’s academic collaborators, researchers from the Novartis Institutes for BioMedical Research in Basel, Switzerland participated in that work.

In previous studies, the kynurenine pathway (KP) of tryptophan degradation has been linked to such neurodegenerative diseases as Huntington’s disease (HD) and Alzheimer’s disease (AD). The kynurenine pathway (KP) is the most important pathway for degradation of the amino acid tryptophan in humans. Patients with HD and AD have elevated levels of two metabolites in the KP–quinolinic acid (QUIN) and 3-hydroxykynurenine (3-HK)–in their blood and brains. Studies in rodents have implicated both of these metabolites in pathophysiological processes in the brain. QUIN, which is a selective N-methyl-D-aspartate (NMDA) agonist, has been implicated in excitotoxicity, which is a mechanism by which excessive stimulation of glutamate receptors causes neuronal dysfunction and cell death. (NMDA receptors constitute a major type of glutamate receptor.) 3-HK is a free radical generator that can mediate neuronal cell death. Intrastriatal injection of QUIN in experimental animals duplicated many of the pathological features of HD. Administration of QUIN to other areas of the brain of experimental animals also duplicated features of AD, such as destruction of  basal forebrain cholinergic neurons projecting to the cortex and memory deficits.

In contrast, kynurenic acid (KYNA), which is formed in a side arm of the KP by conversion of kynurenine by the enzyme kynurenine aminotransferase, appears to be neuroprotective. KYNA is an antagonist of ionotropic excitatory amino acid receptors. In particular, KYNA blocks the neuropathological effects of QUIN. Kynurenine aminotransferase is found in the brain, and is thus capable of transforming kynurenine (which is actively transported into the brain by a neutral amino acid transporter) to KYNA in that organ. The concentration of brain KYNA is often decreased in HD and AD.

All of these studies in rodents were done in the 1980s or 1990s. However, no therapies based on that research have yet been advanced into the clinic.

Studies with JM6, a prodrug small-molecule inhibitor of kynurenine 3-monooxygenase, in wild type mice

In the Zwilling et al. study, researchers studied inhibition of  kynurenine 3-monooxygenase (KMO) as a strategy for inducing a more favorable ratio of KYNA to QUIN in vivo. KMO is the enzyme in the KP that converts kynurenine to 3-hydroxykynurenine, which is further converted in three steps to QUIN. KMO is found at high levels in peripheral blood macrophages and other immune cells in the blood. Inhibition of  KMO results in elevation of kynurenine levels in the blood. This kynurenine can then enter the CNS, where it is converted to the neuroprotective metabolite KYNA.

In 1996, researchers at Roche published the synthesis and characterization of a KMO inhibitor, 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulfonamide, known as Ro 61-8048. Subsequent studies showed that Ro 61-8048 was neuroprotective in rodent models of brain ischemia and cerebral malaria, and in a model of levodopa-induced dyskinesias (movement disorders) in parkinsonian monkeys.

However, Zwilling et al found that Ro 61-8048 was metabolically unstable. They therefore developed an orally bioavailable “slow-release” prodrug of Ro 61-8048, 2-(3,4-dimethoxybenzenesulfonylamino)-4-(3-nitrophenyl)-5-(piperidin-1-yl)methylthiazole (JM6). JM6 was designed to be converted to Ro 61-8048 in the gut. However, when JM6 was administered orally to wild type mice, the researchers found high levels of both JM6 and Ro 61-8048 in the blood. However, neither JM6 nor Ro 61-8048 accumulated to any great extent in the brain, and the brain concentration of both drugs was insufficient to inhibit KMO. Thus neither JM6 nor Ro 61-8048 appeared to cross the blood-brain barrier.

Despite the failure of JM6 and Ro 61-8048 to cross the blood-brain barrier, oral administration of JM6 results in increased brain levels of KYNA. Administration of an inhibitor of kynurenine aminotransferase to the brain inhibits the increase in levels of KYNA in that organ. This is consistent with the hypothesis that Ro 61-8048 inhibition of KMO in the blood results in elevated blood levels of kynurenine, which is transported into the brain. Kynurenine aminotransferase in the brain converts the kynurenine to KYNA. In addition, elevation of KNYA levels in the brain coincides with a decrease in extracellular concentrations of brain glutamate. This is consistent with earlier studies that showed that increases in brain KYNA, via inhibition of presynaptic α7 nicotinic receptors, reduce extracellular brain glutamate levels. Blocking of presynaptic α7 nicotinic receptors results in inhibition of glutamate release from neurons that bear these receptors. Reduction in extracellular brain glutamate levels may be responsible for KYNA’s neuroprotective effects, via reduction of excitotoxicity. However, at high local concentrations of KYNA, this metabolite may also block glutamate receptors directly.

Studies with JM6 in mouse models of Alzheimer’s and Huntington’s disease

After performing these studies in wild type mice, the researchers then tested the effects of JM6 in mouse models of AD and HD. Transgenic J20 mice that express a mutant form of the human amyloid precursor protein (hAPP) develop spatial memory deficits and synaptic loss starting at 4-5 months of age. Oral administration of JM6 starting at 2 months of age gave significant improvement in spatial memory in mice tested at 6 months of age, as compared to untreated J20 APPtg (transgenic APP) mice. JM6 treatment also prevented synaptic loss in J20 APPtg mice. However, JM6 treatment had no effect on beta amyloid (Aβ) plaque load, which was increased in the hippocampus and cortex of JM6-treated and untreated J20 mice. Under the amyloid hypothesis of AD pathogenesis, Aβ plaques are central to the causation of AD.

J20 APPtg mice had lower brain KYNA levels than wild type littermate controls, consistent with findings in AD patients. Treatment of J20 APPtg mice with oral JM6 (over a 120 day period) increased brain and plasma levels of KYNA. KMO activity, and 3-HK and QUIN levels in the brains and QUIN levels in the plasma of J20 APPtg mice treated with JM6 were not significantly different from levels in wild type controls.

The researchers also tested the effects of oral JM6 administration in R6/2 mice, the best characterized mouse model of HD. HD is a trinucleotide repeat disorder in which a cytosine-adenine-guanine (CAG) repeat segment in exon one of the huntingtin gene (HTT) (encoded in the germline of the individual) exceeds a normal range. The HD CAG repeat region encodes 36 or greater repeated glutamines in the polyQ region of the huntingtin protein (Htt); people without the disease have fewer than 36 glutamines. The disease-associated huntingtin protein is neurotoxic.

In the R6/2 mouse model, mice are transgenic for the 5′ end of the human HD gene carrying a large CAG repeat expansion. These mice develop a progressive neurologic disease, including motor deficits, weight loss, and premature death. The researchers started oral JM6 administration at 4 weeks of age, which is an early symptomatic stage in R6/2 mice. JM6 administration had a dramatic dose-dependent effect on survival. JM6 treatment did not affect the weight of the mice, but it did modestly improve performance on an accelerating rotarod (a measure of motor performance). JM6 treatment also prevented synaptic loss and reduced CNS inflammation in R6/2 mice.

JM6 treatment of R6/2 mice did not influence the size or abundance of neuronal inclusion bodies in these mice. These inclusion bodes are related to those seen in HD in humans. Thus in mouse models of both AD and HD, JM6 treatment did not affect the aggregated proteins (Aβ plaques and mutated Htt inclusion bodies, respectively) that are thought to cause the diseases; nevertheless, they ameliorated disease symptoms.

In chronically JM6-treated J20 APPtg (AD model) and R6/2 (HD model) mice, although JM6 and Ro 61-8048 accumulated in plasma, brain levels of these compounds were nil. Thus JM6 treatment of both neurodegenerative disease models resulted in increased brain levels of KYNA and neurodegenerative disease amelioration, despite the inability of JM6 and R0 61-8048 to cross the blood-brain barrier.

JM6 treatment is a surprising therapeutic strategy for neurodegenerative diseases for three reasons.

  • JM6 cannot cross the blood-brain barrier, which is almost always a sine qua non of CNS disease therapy.
  • JM6 ameliorates disease without affecting the protein aggregates that are usually thought to cause the diseases.
  • JM6 ameliorates multiple neurodegenerative diseases.


How might this novel therapeutic strategy be moved into the clinic?

Clinical trials in AD are notoriously long and expensive. Therefore, Drs. Reinhart and Kelly in their Preview suggest that it might be best to first conduct clinical trials in HD, since the cause of HD is much better understood than for AD, and disease progression in placebo controls is better characterized than for AD.

The results of the mouse model studies suggest that JM6 will ameliorate, but not cure, HD and AD. However, since there are no disease-modifying therapies for either disease, demonstrating amelioration of HD comparable to that seen in the mouse models (provided the drug is proven to be safe in humans) will almost certainly gain approval for the drug. However, in the long run JM6 would need to be combined with other disease-modifying drugs to more effectively treat diseases such as HD and AD. Since other drugs  developed for neurodegenerative diseases will almost always act directly in the brain, combining them with JM6, which does not enter the brain, may help maximize the clinical benefit of a combination therapy. It may also aid in minimizing the toxicity of combination therapies (since the two drugs would not interact in the brain).

Lennart Mucke, MD, the director of the Gladstone Institute, suggested that Dr. Muchowski and his colleagues might begin testing JM6 in patients within the next two years.

The ability of JM6/Ro 61-8048 to ameliorate neurodegenerative diseases in animal models also raises questions as to the mechanisms by which it does so, and how these mechanisms might interact with mechanisms thought to be central to the pathobiology of neurodegenerative diseases (e.g., the amyloid and Tau pathways in AD, huntingtin inclusions, inflammatory pathways, apoptotic pathways, etc.). What are the molecular mechanisms downstream from KYNA elevation? Is JM6 treatment prophylactic, or is it efficacious in animals (and in humans) that are already suffering disease symptoms and that have pathogenic protein aggregates?

Research to answer these questions may lead to still newer therapeutic strategies, including potentially more effective combination therapies for neurodegenerative diseases that include JM6.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.


I will lead a workshop entitled “Developing Improved Animal Models in Oncology and CNS Diseases to Increase Drug Discovery and Development Capabilities” at the World Drug Targets Summit in Cambridge MA in July 2011.

Workshops will be held on July 19, and the main conference on July 20-21. I am planning to attend the entire conference.

Our workshop will be a discussion of 2-3 case studies involving development of novel animal models in oncology and CNS diseases, aimed at more closely modeling human disease than current models. Drug discovery and development in these therapeutic areas has been severely hampered by animal models that are  poorly predictive of efficacy. This is a major cause of clinical attrition in these areas.

We shall discuss the implications of these case studies for developing novel therapeutic strategies, target identification and validation, drug discovery, preclinical studies, and reducing clinical attrition. We shall also discuss hurdles to industry adoption of novel animal models developed in academic laboratories.

The main conference will focus on ways of building successful target strategies to minimize drug attrition in the clinic, and specifically how to identify and validate targets that can lead to commercially differentiated products. Speakers will include target discovery and validation leaders from such companies as Pfizer, Merck, NeurAxon, Gilead Sciences, Boehringer Ingelheim, Merrimack Pharmaceuticals, Bayer Schering Pharma AG, FORMA Therapeutics, Roche, Novartis, Tempero Pharmaceuticals, UCB Pharma, Infinity Pharmaceuticals, and from such academic institutions as Harvard Medical School.

The conference agenda and brochure, as well as online registration, are available on the conference website.

Source: Mnolf

In our November 11, 2010 blog post, we discussed the September 2010 acquisition of Seattle biotech firm ZymoGenetics by Bristol-Myers Squibb (BMS). Also in November 2010, Nature Biotechnology published an article about this acquisition, in which I was quoted.

As our blog post states, most commentators believe that BMS’ main motivation for acquiring ZymoGenetics was to gain full ownership of ZymoGenetics’ pegylated interferon-lambda (Peg-IFN-λ) program for treatment of hepatitis C (HepC). The two companies had been been collaborating to develop Peg-IFN-λ since January 2009. However, ZymoGenetics was much more than a one-product company. Its other pipeline drugs included interleukin-21 (denenicokin) for treatment of metastatic melanoma, which is now in Phase 2b development.  And over the years, ZymoGenetics has proven to be an important drug discovery engine, from the days in which it was a division of Novo Nordisk, and continuing on into 2010.

Now–as of the first week of January 2011–we learn that former ZymoGenetics CEO Douglas E. Williams Ph.D. has been named as Executive Vice President, R&D., at Biogen Idec (Weston, MA).

Dr. Williams has over 20 years of biotech R&D and senior leadership experience. He was the chief technology officer at Seattle biotech firm Immunex, and played a significant role in the discovery and development of the blockbuster tumor necrosis factor (TNF) inhibitor etanercept (Enbrel). After Amgen’s 2002 acquisition of Immunex, Dr. Williams resigned from Amgen later in 2002, and moved on to Seattle Genetics in 2003 as chief scientific officer (CSO). In 2004, he joined ZymoGenetics as chief scientific officer (CSO). On January 1, 2009, he became ZymoGenetics’ CEO.

During his tenure as CSO and then CEO of ZymoGenetics, the company achieved considerable success in the development of its pipeline products, especially Peg-IFN-λ and  interleukin-21. And the company entered into its $1.1 billion agreement with BMS to codevelop Peg-IFN-λ. However, during Dr. Williams’ tenure as CEO, ZymoGenetics had some financial rough spots, mainly caused by the lack of commercial success of the company’s first self-marketed product, recombinant thrombin (Recothrom). This was compounded by failed clinical trials of the company’s immunomodulatory drug atacicept, which is now being developed by Merck Serono. After a series of downsizing moves, ZymoGenetics agreed to be acquired by BMS in October 2010. In November 2010, Dr. Williams left ZymoGenetics and became a “free agent”, followed by his joining Biogen Idec in January 2011.

Biogen Idec, which was founded as Biogen in 1978 and merged to form Biogen Idec in 2003, is one of the world’s major biotech companies, and has long been a major fixture of the Boston-Cambridge biotech scene. The company had 2009 revenue of $4.38 billion. However, Biogen Idec had some ups and downs of its own in recent years. It has been targeted for reorganization, breakup, or sale by activist investor Carl Icahn, who currently owns 5.4% of the company’s shares, and who controls three seats on Biogen Idec’s board as the result of  series of proxy fights.

During 2010, long-time CEO (and Icahn target) James Mullen retired from the company, and was succeeded by former Exelixis (South San Francisco, CA) CEO George Scangos, Ph.D. In January 2011, at the same time as Dr. Williams joined Biogen Idec, the company announced that Steven H Holtzman (who was formerly the CEO of Cambridge MA biotech Infinity Pharmaceuticals) would be executive vice president of corporate development.

Biogen Idec derives most of its revenues from three drugs–multiple sclerosis (MS) treatments Avonex (interferon beta-1a) and Tysabri (natalizumab), and Rituxan (rituximab), a treatment for non-Hodgkin’s lymphoma. Tysabri is also approved for treatment of Crohn’s disease and is co-marketed with Élan, and Rituxan is also approved for rheumatoid arthritis and is co-marketed with Roche/Genentech.

Among these products, Avonex (which was introduced in 1996, and is Biogen Idec’s largest selling drug) and Rituxan are maturing. In particular, Avonex faces increased competition from newer products. Growth in sales and revenues from these two products is slowing.

Tysabri is intended to be Biogen Idec’s growth driver. However, Tysabri has had major issues. Soon after its launch in 2004, Biogen Idec withdrew Tysabri from the market, after it was linked with three cases of the rare neurological condition progressive multifocal leukoencephalopathy (PML), when co-administered with Avonex.  PML is caused by the JC virus, which is normally controlled by he immune system, but which can rarely cause disease in patients under immunosuppresive therapies such as the Tisabri/Avonex combination. After a safety review and no further deaths, the drug was returned to the US market in 2006 under a special prescription program, in part as the result of pleas by MS patients. However, since then additional cases of PML–including fatalities–have occurred.

In December 2010, Biogen Idec and Elan submitted a supplemental Biologics License Application (sBLA) to the FDA and a Type II Variation to the European Medicines Agency (EMA), proposing updated product labeling to include anti-JC virus antibody status. The companies propose using this test to help stratify the risk of developing PML in patients treated with Tysabri. Biogen Idec expects that a commercial anti-JC virus antibody test will be available later in 2011. It is expected that this test will help to lower the risk of Tysabri-associated PML, which is low to begin with.

In addition, Tysabri faces potential strong competition from the first approved oral treatment for MS, fingolimod (Novartis’ Gilenya), which the FDA approved in September 2010. The day that Gilenya was approved, Biogen Idec issued a press release acknowledging the desire of MS patients for an oral treatment, and noting that it also has an oral MS treatment in Phase 3 trials, BG-12.

Biogen Idec estimated that as of the end of 2010, approximately 56,600 MS patients were using Tysabri worldwide. That represented an increase of 1,700 patients in the fourth quarter and 8,200 patients over the course of 2010.

In November 2010, Dr. Scangos announced a reorganization of Biogen Idec. As of that date, the company would focus on neurology, and leverage its strengths in biologics research and development (R&D) and manufacturing to pursue select, high-impact biologic therapies and to be a leading collaborator in the biotechnology industry. (Biogen Idec’s efforts in biologics might, for example, include entering the biosimilars market.) Biogen Idec also terminated its efforts in cardiovascular medicine, and is seeking to spin out or outlicense its oncology programs.

The restructuring also involved consolidating its sites, and reducing its work force by 13%, or 650 full-time positions. As a result of the restructuring, the company expected to save approximately $300 million annually. Dr. Scangos said that the restructuring would enable Biogen Idec to gain focus and to become more nimble.

The company intends to become a global leader in neurological diseases. This will involve not only maximizing the potential of its two marketed MS drugs, but also bringing forward its MS pipeline products. Biogen Idec will also pursue programs in amyotrophic lateral sclerosis (ALS)/Lou Gehrig’s disease and Parkinson’s disease.

Biogen Idec’s late-stage products in neurology are shown in the table. (Please click on the table to read it clearly.) The company intends to launch five new products by 2015.

Source: Haberman Associates

Although Biogen Idec now has several late-stage products moving toward commercialization, the company’s R&D productivity has lagged in recent years. The company has not launched a new drug since Tysabri was approved in 2004. Dr. Williams says that he is planning a review of he company’s R&D organization and its pipeline. He intends especially to focus on Biogen Idec’s early- and mid-stage programs. Dr. Williams intends to boost these programs both via internal R&D and via licensing and acquisition to bring in externally developed compounds.

Overall, Dr. Williams hopes to return Biogen Idec to the culture of a biotech start-up. “We don’t have the luxury of sitting back. We have to push hard like we are a scrappy, hungry, cash-starved biotech,” he says. Dr. Williams’ statement is in accord with that of Dr. Scangos, who speaking at the J.P. Morgan 29th Annual Healthcare Conference in January 2011, said that Biogen Idec had the choice of being either a small pharma or a big biotech. The company has chosen to be a big biotech.

We wish Dr. Williams–working together with George Scangos and Steven Holtzman–well as they work to return Biogen Idec to productive and innovative R&D.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

In Chapter 7 of our March 2010 book-length report, Animal Models for Therapeutic Strategies (published by Cambridge Healthtech Institute), we discussed recently-developed methods for producing knockout rats. These methods included zinc-finger nuclease (ZFN) genome editing and transposon mutagenesis in cultured spermatogonial stem cells. Our most extensive discussion was of the ZFN editing technology, which was developed by Sangamo BioSciences (Richmond, CA), and is the basis of the knockout rat models marketed by Sigma-Aldrich Advanced Genetic Engineering (SAGE). We also mentioned the SAGE knockout rat platform in an earlier blog post.

In Chapter 7 of our report, we also mentioned that it would now also be possible to construct knockout rats “the good old way”–using the same homologous recombination technology that researchers use to create knockout mice. Drs. Mario R. Capecchi, Martin J. Evans and Oliver Smithies were awarded the Nobel Prize in Physiology or Medicine for 2007 for having developed this technology in the late 1980s. To construct knockout mice, researchers isolate and culture mouse embryonic stem (ES) cells. These are derived from the inner cell masses of preimplantation mouse blastocyst embryos, and grown under particular culture conditions. These cells are subjected to homologous recombination with a vector containing a truncated version of the gene to be targeted, to eventually yield knockout mouse strains.

It has not been possible to develop knockout rats because the conditions for culturing ES cells worked only for a few inbred mouse strains, and not at all for either most mouse strains or for the rat. Conditions for culturing mouse ES cells are complex. They involve the use of feeder fibroblasts and/or the cytokine leukemia inhibitory factor (LIF), together with selected batches of fetal calf serum or bone morphogenetic protein (BMP). These culture conditions had been determined empirically.

In 2008, Dr. Austin Smith (Director of the Wellcome Trust Centre for Stem Cell Research, University of Cambridge [Cambridge, UK]) and his colleagues developed culture conditions that allowed them to culture rat ES cells that were capable of transmitting their genomes to offspring. These ES cells could also be used to produce knockout rats.

Dr. Smith and his colleagues realized that the standard conditions for culturing mouse ES cells expose the cells to inductive stimuli (e.g., fibroblast growth factor 4 [FGF4]), which can activate ES cell commitment and differentiation. The aim of ES cell culture is to expand the cell population while maintaining pluripotency.  The researchers therefore cultured rat ES cells with leukemia inhibitory factor (LIF)-expressing mouse fibroblast feeder cells, in a medium containing two or three small-molecule inhibitors of pathways involved in ES cell commitment and differentiation, plus human LIF. (LIF supports proliferation of ES cells in an undifferentiated state.) This medium is known as 2i (for 2-inhibitors) or 3i medium.

Rat ES cells cultured in this manner expressed key molecular markers found in mouse ES cells. They also, when injected into blastocysts, can give rise to chimeric rats; i.e., they transmute their genomes into offspring. Such cultured rat ES cells thus are capable of being used to construct knockout rats.

In the 9 September 2010 issue of Nature, Dr. Qi-Long Ying (University of Southern California, Los Angeles CA) and his colleagues published the first study describing construction of a knockout rat strain via homologous recombination. (Dr. Ying, then at the University of Edinburgh, had been on the team led by Austin Smith that developed culture methods for rat ES cells.) This rat strain is a p53 gene knockout. The researchers designed a targeting vector to disrupt the p53 tumor suppressor gene via homologous recombination; the vector allowed for antibiotic selection for cells that had been successfully targeted. They transfected this vector into rat ES cells cultured in 2i medium, performed the antibiotic selection, and cultured the resistant cells. These cells were shown to have one of their two (since they were diploid) p53 genes disrupted. The researchers were able to routinely generate p53-targeted rat ES cells by this method. They also injected p53-targeted rat ES cells into rat blastocysts, transferred the blastocysts into pseudo-pregnant female rats, and obtained chimeric offspring. However, in the first studies, the p53-targeted rat ES cells exhibited low germline transmission efficiency.

In the mouse system, the failure of cultured ES cells to contribute to the germline is often caused by chromosomal abnormalities in the ES cells. This was also the case with the rat ES cells. In the case of mouse ES cell culture, cells with chromosomal abnormalities have a selective growth advantage over those with normal karyotypes. The smaller, slower-growing mouse ES cell clones tend to have normal karyotypes, and to give improved germline transmission. The researchers therefore subcloned their p53 gene-targeted rat ES cells, and selected for small, slower-growing subclones. These rat ES cell subclones were euploid. When injected into blastocysts, these rat ES cell clones gave rise to chimeric rats that the researchers further bred to generate homozygous p53 gene-targeted (i.e., p53 knockout, or p53 homozygous null) rats.

Using these methods, it should be possible to generate knockout rats for other genes routinely, including sophisticated knockouts such as tissue-specific gene knockouts.

Meanwhile, SAGE has generated p53 knockout rats, using its ZFN technology. As with the original p53 knockout mice, these rats develop normally, but are prone to development of spontaneous tumors. p53 knockout rats generated via homologous recombination should also be susceptible to spontaneous generation of tumors. However, as yet no data has been published. It remains to be seen which of these systems–p53 knockout mice or p53-knockout rats generated via either homologous recombination or ZFN editing, will be most useful in basic cancer research, or in such applications as carcinogenicity screening of compounds.

Why is the ability of researchers to generate knockout rats, as opposed to knockout mice, so important? The anatomy and physiology of the rat is closer to humans than is the mouse. There are also many rat models of complex human diseases (especially cardiovascular and metabolic diseases) that are better disease models than those based on inbred mouse strains. In addition, the larger size of the rat facilitates experimental procedures that involve surgery, getting blood samples for analysis, or isolation of specific cell populations. Researchers usually prefer rats over mice for physiological and nutritional studies, studies of psychiatric diseases, and in cases when a particular rat disease model is more applicable to a project than mouse strains. The rat is also widely used in preclinical efficacy and safety studies.

With respect to models for central nervous system (CNS) diseases, gene-targeted and transgenic rat models may be expected to be better than mouse models. The rat is more intelligent than the mouse, and has a bigger brain. Unlike mice, rats are sociable and easily trained. Moreover, there are some new rat models of cognition, which enable researchers to perform studies that they previously thought could only be done in nonhuman primates. And optogenetics technology, which allows researchers to engineer specific neurons so that their activity can be switched on or off with laser light, in order to dissect the role of these neurons in behavior, is being implemented in rats. These new developments, together with knockout and transgenic technologies, should allow researchers to develop new rat models of psychiatric diseases, as well as of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. The lack of good animal models is a major factor in the high clinical attrition rate of CNS drugs, so new models are needed. There are of course no guarantees that novel rat models will help lower CNS drug attrition rates, but it is well worth trying these new approaches.

As we also discussed in Chapter 7 of Animal Models for Therapeutic Strategies, researchers are also interested in developing animal models based on mammalian species other than the mouse and the rat. We discussed methods for gene targeting by recombinant adeno-associated virus (rAAV) in pigs and ferrets in that chapter. In principle, ZFN editing technology could be also used to generate gene knockouts in mammalian species other than rodents. Moreover, the type of research done in the rat by Austin Smith, Qi-Long Ying, and their colleagues might be applied to developing culture conditions for ES cells of other mammalian species, which could set the stage for developing gene knockouts in these species via homologous recombination.

In the December 15, 2009 issue of Neurology, a research report by Stephen Salloway and his colleagues at the Butler Hospital and Brown University (Providence, RI) and an editorial by Dan Kaufer and Sam Gandy (University of North Carolina at Chapel Hill) focus on a Phase II multicenter placebo-controlled clinical trial of Elan/Wyeth’s bapineuzumab (AAB-001) in patients with mild to moderate Alzheimer’s disease (AD). (Wyeth is now part of Pfizer.) (A subscription is required to read the full text of both of these articles.) Bapineuzumab is a monoclonal antibody (MAb) drug that is specific for amyloid-β (Aβ) peptide. The dominant paradigm among AD researchers and drug developers is that the disease is caused by aberrant metabolism of Aβ, resulting in accumulation of neurotoxic Aβ plaques. This paradigm is known as the “amyloid hypothesis”.

The overall result of the study by Salloway et al. was that there was no difference in cognitive function between patients in the drug-treated and the placebo groups. However, the study did not have sufficient statistical power to exclude the possibility that there was such a difference. About 10% of patients treated with the agent also experienced vasogenic edema (VE), which was reversible. (Cerebral VE is the infiltration of intravascular fluid and proteins into brain tissue, as the result of breakdown of the blood-brain barrier.)

Retrospective analysis of the data suggested that bapineuzumab-treated patients who were not carriers of the apolipoprotein E epsilon4 allele (ApoE4) showed improved cognitive function as compared to placebo treatment, and that they had a lower incidence of VE than ApoE4 carriers. The ApoE4 polymorphism is the only known, well-characterized genetic risk factor associated with the development of late-onset AD. Of the three common isoforms of ApoE, ApoE3 is the most common, followed by ApoE4 and ApoE2, respectively. Unlike ApoE4, the ApoE2 allele appears to protect against development of AD. Some researchers estimate that allelic variations in ApoE may account for over 95% of AD cases.

In the study by Salloway et al., nearly two-thirds of the AD patients carried one or more ApoE4 alleles; thus only the remaining one-third of patients appeared to show positive effects of bapineuzumab treatment according to the retrospective analysis. However, the idea that the drug is efficacious in ApoE4 noncarriers is only a hypothesis, which will require prospective clinical trials to confirm. Elan and Pfizer are now conducting large Phase III clinical trials of bapineuzumab, which have prospectively segregated enrollment into ApoE4 carrier and noncarrier groups.

The hypothesized association of ApoE4 noncarrier status of AD patients with bapineuzumab efficacy and safety has been used as a case study in workshops on stratified medicine sponsored by the FDA, MIT, and industry partners in 2009 and 2010. You can read about the October 2009 workshop here. The most recent workshop was held at MIT on January 19, 2010. In these workshops, two case studies were discussed: the use of diagnostic tests for the HER2 receptor in identifying breast cancer patients who are likely to benefit from treatment with trastuzumab (Genentech/Roche’s Herceptin), and the bapineuzumab/ApoE4 case. The HER2/ trastuzumab relationship is well known and well characterized, and is considered to be a paradigm of stratified medicine. This contrasts with the bapineuzumab/ApoE4 association, which remains a hypothesis pending the results of the Phase III prospective clinical studies.

A growing minority of researchers is skeptical that the amyloid hypothesis is sufficient to account for AD pathogenesis in all stages of the disease or in various disease subpopulations, and they are investigating other pathways that may contribute to the disease, either in combination with the amyloid pathway or as alternative mechanisms. We have discussed alternative hypotheses for AD pathogenesis in a 2004 article published in Genetic Engineering News (available on our website), and in book-length reports published by Cambridge Healthtech Institute in 2006 and in 2009.

The search for alternative hypotheses takes on added urgency because of the clinical failure of several AD drugs that had been designed based on the amyloid hypothesis. These include Neurochem’s (now Bellus Health) Alzhemed (3-amino-1-propanesulfonic acid) and Myriad Pharmaceuticals’ Flurizan (tarenflurbil), both of which failed in Phase III clinical trials. Based on the overall results of the Phase II trial of bapineuzumab, most researchers and industry commentators would add bapineuzumab to the list, unless the stratified Phase III trial shows that the drug is significantly efficacious and safe for ApoE4 noncarriers.

Since ApoE4 carrier status is such a prominent risk factor for developing late-onset AD, might ApoE4 itself be a target for drug discovery in AD? Drs. Kaufer and Gandy suggest that such an approach might be fruitful, whatever the outcome of the Phase III trial of bapineuzumab. Several academic laboratories have been investigating mechanisms by which ApoE4 may be involved in the pathobiology of AD. You may read two recent papers on this subject here and here. ApoE4 may contribute to AD pathogenesis via multiple mechanisms, including by causing synaptic deficits and mitochondrial dysfunction in neurons, and by inducing endoplasmic reticulum stress leading to astrocyte dysfunction.

Given the prominence of ApoE4 expression as a risk factor for AD, the study of the mechanistic basis of ApoE4’s role in AD pathobiology needs greater attention. Hopefully, this research will lead to the development of novel therapeutic strategies for AD.