Animal Models

Identification of a novel Alzheimer’s disease pathway provides potential new avenues for drug discovery

Published by Allan Haberman on January 9, 2013 | Leave a response

Neurofibrillary tangle.

In August and September of 2012, we published three articles on Alzheimer’s disease on the Biopharmconsortium Blog:

New genetics study supports the amyloid hypothesis of Alzheimer’s disease–but the drugs still don’t work! (August 19, 2012.)
Here we go again–Lilly’s Alzheimer’s drug solanezumab fails to show efficacy in Phase 3, but company is “encouraged” by secondary analysis. (August 28, 2012.)
Alzheimer’s disease–where do we go from here? (September 20, 2012.)

Subsequent to the publication of our articles–on 21 November, 2012–the Wellcome Trust announced the identification of a novel pathway involved in the pathogenesis of Alzheimer’s disease (AD). This research was led by Professor Simon Lovestone and Dr Richard Killick (Kings College, London U.K.), and was published in the online edition of Molecular Psychiatry on 20 November 2012. The Wellcome Trust helped to fund the research.

As we have discussed in earlier articles on this blog, the dominant paradigm among AD researchers and drug developers is that the disease is caused by aberrant metabolism of amyloid-β (Aβ) peptide, resulting in accumulation of neurotoxic Aβ plaques. This paradigm is known as the “amyloid hypothesis”. AD is also associated with neurofibrillary tangles (NFTs) which are intracellular aggregates of hyperphosphorylated tau protein. In contrast to the amyloid hypothesis, some AD researchers have postulated that NFT formation is the true cause of AD. The new research links amyloid toxicity to the formation of NFTs, and identifies potential new drug targets.

The new study is based on the discovery of the role of clusterin–an extracellular chaperone protein–in sporadic (i.e., late-onset, non-familial) AD. The gene for clusterin, CLU, has been identified as a genetic risk factor for sporadic AD via a genome-wide association study published in 2009. Clusterin protein levels are also increased in the brains of transgenic mouse models of AD that express mutant forms of amyloid precursor protein (APP), as well as in the serum of humans with early stage AD.

The researchers first studied the relationship between Aβ and clusterin in mouse neuronal cells in culture. Aβ rapidly increases intracellular concentrations of clusterin in these cells. Aβ-induced increases in clusterin drives transcription of a set of genes that are involved in the induction of tau phosphorylation and of Aβ-mediated neurotoxicity. This pathway is dependent on the action of a protein known as Dickkopf-1 (Dkk1), which is an antagonist of the cell-surface signaling protein wnt. The transcriptional effects of Aβ, clusterin, and Dkk1 are mediated by activation of the wnt-planar cell polarity (PCP) pathway. Among the target genes in the clusterin-induced DKK1-WNT pathway that were identified by the researchers are EGR1 (early growth response-1), KLF10 (Krüppel-like factor-10) and NAB2 (Ngfi-A-binding protein-2)–all of these are transcriptional regulators. These genes are necessary mediators of Aβ-driven neurotoxicity and tau phosphorylation.

The researchers went on to show that transgenic mice that express mutant amyloid display the transcriptional signature of the DKK1-WNT pathway, in an age-dependent manner, as do postmortem human AD and Down syndrome hippocampus. (Most people with Down syndrome who survive into their 40s or 50s suffer from AD.) However, animal models of non-AD tauopathies (non-AD neurodegenerative diseases associated with pathological aggregation of tau, and formation of NFTs, but no amyloid plaques) do not display upregulation of transcription of genes involved in the DKK1-WNT pathway, nor does postmortem brain tissue of humans with these diseases.

The Kings College London researchers concluded that the clusterin-induced DKK1-WNT pathway may be involved in the pathogenesis of AD in humans. They also hypothesize that such strategies as blocking the effect of Aβ on clusterin or blocking the ability of Dkk1 to drive Wnt–PCP signaling might be fruitful avenues for AD drug discovery. According to the Wellcome Trust’s 21 November 2012 press release, Professor Lovestone and his colleagues have shown that they can block the toxic effects of amyloid by inhibiting DKK1-WNT signaling in cultured neuronal cells. Based on these studies, the researchers have begun a drug discovery program, and are at a stage where potential compounds are coming back to them for further testing.

________________________________

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 an initial one-to-one consultation on an issue that is key to your company’s success, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Posted in Animal Models, Anti-Aging, Drug discovery, Neurodegenerative diseases

Alzheimer’s disease–where do we go from here?

Published by Allan Haberman on September 20, 2012 | Leave a response

New Alzheimer’s disease model, the CVN mouse

Our August 19, 2012 and our August 28, 2012 articles on this blog focused on the latest developments in Alzheimer’s disease (AD) drug development. To summarize the conclusions of the articles:

The results of a new genetic study by DeCode Genetics and its collaborators strongly support the amyloid hypothesis of AD, and especially the hypothesis that reducing the β-cleavage of APP [e.g., by use of an inhibitor of β-secretase (also known as the β-site APP cleaving enzyme 1, or BACE1)] may protect against the disease.

Nevertheless, in Phase 3 trials of two anti-amyloid monoclonal antibody (MAb) drugs in patients with mild to moderate AD–Pfizer/Janssen’s bapineuzumab (often called “bapi” for short) and Lilly’s solanezumab–the drugs failed their primary cognitive and functional endpoints.

Roche/Genentech, as well as two academic consortia, have begun clinical trials of anti-amyloid MAb drugs in asymptomatic patients with mutations that predispose them to develop AD, or in asymptomatic patients with amyloid accumulation. These studies are based on the hypothesis that the reason for the failure of anti-amyloid MAb drugs in clinical trials has been that the patients being treated had suffered extensive, irreversible brain damage. Treating patients at a much earlier stage of disease with these agents might therefore be expected to be more successful.

Analyses of the data from the Phase 3 studies of both bapi and solanezumab will be presented in scientific meetings in October 2012. An academic research consortium will present its independent analysis of the data from the EXPEDITION studies of solanezumab at the American Neurological Association (ANA) meeting in Boston on October 8, 2012, and at the Clinical Trials on Alzheimer’s Disease (CTAD) meeting in Monte Carlo, Monaco, on October 30, 2012.

According to a September 11, 2012 news article in Drug Discovery & Development, researchers who conducted the Phase 3 trials of bapi found evidence that the drug stabilized amyloid plaque in the brain and may have ameliorated further nerve damage in patients treated with the drug. This finding is among the results to be presented in the October meetings.

Development of BACE1 inhibitors

Strictly speaking, the results of the DeCode Genetics study most strongly support the development of BACE1 inhibitors. In our August 28, 2012 article, we link to a 2010 review that includes a discussion of companies developing BACE1 inhibitors. However, we also note that the development of BACE1 inhibitors has been elusive. This is because of medicinal chemistry considerations. Specifically, it has been difficult to design a specific, high-affinity inhibitor of the BACE1 active site that can cross the blood-brain barrier and which has good drug-like ADME (absorption, distribution, metabolism and excretion) properties. Nevertheless, recently progress has been made in developing such compounds, and several companies are developing BACE1 inhibitors and have entered them into early-stage clinical trials.

Among the companies developing BACE1 inhibitors, as listed in a recent post on Derek Lowe’s In The Pipeline blog are CoMentis/Astellas, Merck, Lilly, and Takeda.

Satori Pharmaceuticals is developing γ-secretase inhibitors

Developing γ-secretase inhibitors has been abandoned by the vast majority of companies, because of the essential role of these enzymes in the Notch pathway and other pathways involved in normal physiology. As a result, development of γ-secretase inhibitors for AD has not progressed beyond the preclinical stage.

Nevertheless, Satori Pharmaceuticals, a Cambridge, MA venture capital-backed biotech company, is now actively involved in developing γ-secretase inhibitors. Satori’s γ-secretase inhibitors are based on a proprietary scaffold derived from a compound isolated from the black cohosh plant (Actaea racemosa). The company utilized modern synthetic and medicinal chemistry to derive compounds based on this scaffold that they believe are suitable for long-term oral therapy for AD in humans. Satori’s lead compound, SPI-1865, is a potent γ-secretase modulator that decreases levels of the amyloidogenic Aβ42 peptide as well as Aβ38, increases levels of Aβ37 and Aβ39, but does not affect Aβ40. Researchers believe that decreasing Aβ42 levels in favor of shorter, less amyloidgenic A-beta forms is beneficial in treatment of AD. SPI-1865 is also selective for Aβ42 lowering over the inhibition of Notch processing, and appears to be free of any other off-target activities.

In animal models [e.g., wild type mice and rats, and transgenic mice (Tg2576) that overexpress APP and thus have high levels of Aβ peptides] orally-administered SPI-1865 has been found to lower brain Aβ42. SPI-1865 has good brain penetration in these models, and a long half-life that should permit once a day dosing in humans.

SPI-1865 is now in the preclinical stage. Satori plans to file an Investigational New Drug (IND) Application with the FDA in late 2012 with the goal of enabling initial human testing to begin in the early part of 2013.

The overall strategy of Satori Pharmaceuticals is to develop novel and proprietary oral small-molecule drugs that are designed for chronic dosing, and can be used to treat patients with early-stage (or presymptomatic) AD for years or decades. Such a strategy will require compounds that are efficacious and exceptionally safe. Satori expects that its unique γ-secretase inhibitors will have these properties. However, this needs to be shown in human clinical trials. Moreover, this strategy–as with all strategies that involve treating AD in its earliest stages–will involve the development of reliable biomarkers and companion diagnostics.

A new mouse model for AD

As Derek Lowe says in an August 31, 2012 post on “In the Pipeline” with respect to Lilly’s AD drugs, anti-amyloid MAbs, BACE1 inhibitors, and γ-secretase inhibitors are “some of the best ideas that anyone has for Alzheimer’s therapy”. Given the APP processing pathway as illustrated in the figure at the top of our August 28, 2012 article, these are the “sensible” and “logical” alternatives.

Nevertheless, there is the nagging feeling among many AD researchers that we do not understand the causes of AD, especially sporadic AD, which represents around 95% of all cases of the disease. Sporadic AD occurs in aging individuals who have normal genes for the components of the APP processing pathway. Not only do we not understand the pathobiology of sporadic AD, but we have little understanding of the normal physiological function of APP and of APP processing. Processes that may be involved in the initiation of sporadic AD may include not only those involved in Aβ production, but also those involved in Aβ clearance.

An important tool in understanding the pathobiology of AD, and potentially in developing novel therapies for the disease, would be an animal model that recapitulates the human disease as closely as possible. We published an article on AD mouse models that were designed to more closely recapitulate human AD than the most commonly used models in the September 15, 2004 issue of Genetic Engineering News. However, since the publication of our article, Carol A Colton, Ph.D. (Duke University Medical Center, Durham, NC) and her colleagues have published on their research aimed at producing an even better mouse model, known as the CVN mouse. They published their research in two articles, one in PNAS in 2006 and the other in the Journal of Neuroscience in 2008.

Charles River Laboratories (CRL) (Wilmington, MA) now offers the CVN mouse to researchers who might wish to employ it in their AD research. CRL has also recently produced a webinar (with the participation of Dr. Colton) on the CVN mouse, entitled “CVN Mouse: A More Translatable Alzheimer’s Efficacy Model”. You may access this webinar by registering at http://www.criver.com/thesource.

Genome-wide association studies (GWAS) in humans, as well as various functional studies, have implicated variants in genes involved in inflammation and immune responses in susceptibility to late-onset, sporadic AD in humans. The Colton group, noting that commonly-used mouse models of AD recapitulated human disease very poorly, looked for differences between mice and humans in innate immunity. The biggest difference they found was that expression of nitric oxide synthase 2 (NOS2) the inducible form of nitric oxide synthase, is high in mice and low in humans. NOS2 is an enzyme that produces nitric oxide (NO), a highly reactive oxidant that can serve in signal transduction, neurotransmission and in cell killing by macrophages. Microglia, the macrophages of the brain, express NOS2 and NO. The Colton group has been studying the role of microglia and oxidants and antioxidants in microglia that can produce oxidative stress in the brain in normal aging and in AD.

Because of the striking difference in NOS2 expression between mice and humans, the Colton group created a transgenic mouse AD model by crossing mice that expressed a mutant form of human APP known as APPSwDI (APP Swedish Dutch Iowa) with NOS2 knockout (NOS2 -/-) mice. The APPSwDI transgenic mouse, a well-characterized standard AD mouse model, was chosen because it expresses low levels of APP and high levels of Aβ peptides in the brain. The APPSwDI/NOS2 -/- mouse is the CVN mouse that is available from CRL.

Unlike APPSwDI mice and other standard AD mouse models, the CVN mouse recapitulates many features of human AD as the animals age, including AD-like amyloid pathology (starting at 6 weeks of age, which is early), perivascular deposition of amyloid, AD-like tau pathology (including aggregated hyperphosphoryated tau), AD-like neuronal loss, and reduction in interneuron numbers (including NPY interneurons). Age-related cognitive (learning and memory) loss (as assessed by the radial arm water maze test) was also seen. The researchers also saw increases in immune activation and inflammation (e.g., microglial activation) over the course of the disease; this appeared to be dependent on increases in Aβ and in tau.

The researchers also used the mouse to study changes in immune-related proteins over the course of the disease. Several protein that are encoded by genes that have been associated with sporadic AD via GWAS change over time in this mouse model, including APOE (which has been known to be important in AD for a long time) and BIN1. Other proteins that change over the course of disease include the complement component C1QB, and the centrosomal protein ninein. Immune activation genes such as those that encode IL-1α and TGF-β also show changes over the course of disease in these mice. The Colton group will soon publish their work on changes in these proteins and genes in the CVN mouse in a peer-reviewed journal.

In summary, the CVN mouse more faithfully models AD-like progression than other mouse models that have been used to study AD, including those that have been used in preclinical studies of such failed drug candidates as solanezumab, bapineuzumab, Flurizan (tarenflurbil), and Alzhemed (3-amino-1-propanesulfonic acid). It also allows researchers to study the role of genes and proteins such as those identified in GWAS studies in AD, and especially in sporadic AD. (However since the CVN mouse expresses a mutant form of APP, it can not be used to study all aspects of the pathophysiology of sporadic AD, especially the initiation of the disease process.) The CVN mouse can also be used in drug discovery and preclinical studies.

One example of such drug discovery studies is being carried out by the Colton group. They have recently been studying small APOE mimetic peptides in CVN mice. The subcutaneously administered APOE mimetics were reported to significantly improve behavior, while decreasing the inflammatory cytokine IL-6, as well as decreasing neurofibrillary tangle-like and amyloid plaque-like structures. These improvements are associated with apoE mimetic-mediated increases in protein phosphatase 2A (PP2A) activity. [Decreased PP2A levels in AD may be involved in formation of neurofibrillary tangles (NFTs) which are aggregates of hyperphosphorylated tau; PP2A may also be involved in the production of Aβ peptides. The APOE mimetic are thus potential AD therapeutics.

_________________________________________

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.

Posted in Animal Models, Drug development, Drug discovery, Immunology, Neurodegenerative diseases

Co-clinical mouse/human trials for cancer continue to advance

Published by Allan Haberman on May 8, 2012 | Leave a response

RAS/BRAF/PI3K pathways. Source: Source BioScience

Two previous articles on this blog have included discussions of the “co-clinical mouse/human trial” strategy for improving mouse models of human cancer, and simultaneously improving human clinical trials of drugs for these cancers. Now comes an article on the use of a co-clinical trial strategy in personalized treatment of non-small cell lung cancer (NSCLC) in the 29 March 2012 issue of Nature. In the same issue of Nature is a News and Views article by Genentech’s Leisa Johnson Ph.D. that provides a minireview of the research article.

As we discussed in our April 15, 2010 article on this blog, the co-clinical trial strategy has been developed by Pier Paolo Pandolfi, MD, PhD (Director, Cancer and Genetics Program, Beth Israel-Deaconess Medical Center Cancer Center and the Dana-Farber/Harvard Cancer Center) and his colleagues.

As discussed in that article, these researchers constructed genetically engineered transgenic mouse strains that have genetic changes that mimic those found in human cancers. These mouse models spontaneous develop cancers that resemble the corresponding human cancers. In Dr. Pandolfi’s ongoing co-clinical mouse/human trial project, researchers simultaneously treat a genetically engineered mouse model and patients with tumors that exhibit the same set of genetic changes with the same experimental targeted drugs. The goal of this two-year project is to determine to what extent the mouse models are predictive of patient response to therapeutic agents, and of tumor progression and survival. The studies may thus result in validated mouse models that are more predictive of drug efficacy than the currently standard xenograft models.

The human clinical trials being “shadowed” by simultaneous studies in mice included Phase 3 trials of several targeted therapies for lung and prostate cancer. Xenograft models in which tumor tissue from the patients had been transplanted into immunosuppressed mice were also being tested in parallel with the genetically engineered mouse models. This project represents the most rigorous test to date of how well genetically engineered mouse models of cancer can predict clinical outcomes.

Our October 28, 2011 blog article, which is mainly a review of a 29 September 2011 Nature article by Nature writer Heidi Ledford, Ph.D., focuses on ways to fix the clinical trial system. Our article includes a discussion of a co-clinical trial published in January 2011. This trial utilized two genetically-engineered PDGF (platelet-derived growth factor)-driven mouse models of the brain tumor glioblastoma multiforme (GBM), one of which had an intact PTEN gene and the other of which was PTEN deficient. In this trial, researchers tested the Akt inhibitor perifosine (Keryx Biopharmaceuticals, an alkylphospholipid) and the mTOR inhibitor CCI-779 (temsirolimus; Pfizer’s Torisel), both alone and in combination, in vitro and in vivo. The drugs and drug combinations were tested in cultured primary glioma cell cultures derived from the PTEN-null and PTEN-intact mouse PDGF-driven GBM models, and in the animal models themselves.

The studies showed that both in vitro and in vivo, the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null cells in animals was achieved by using both inhibitors in combination. In vivo, the decreased Akt and mTOR signaling seen in mice treated with the combination therapy correlated with decreased tumor cell proliferation and increased cell death; these changes were independent of PTEN status. The co-clinical animal study also suggested new ways of screening GBM patients for inclusion in clinical trials of treatment with perifosine and/or CCI-779.

The new co-clinical trial reported in the March 2012 issue of Nature

The March 2012 Nature report describes research carried out by a large, multi-institution academic consortium, which included Dr. Pandolfi. It focuses on strategies for treatment of patients with non-small-cell lung cancer (NSCLC) with activating mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog). These mutations occur in 20–30% of NSCLC cases, and patients whose tumors carry KRAS driver mutations have a poor prognosis. Moreover, KRAS is a “hard” or “undruggable” target, and no researchers have thus been able to discover inhibitors of oncogenic KRAS.

Because of the intractability of oncogenic KRAS as a target, researchers have been attempting to develop combination therapies for mutant-KRAS tumors (including, for example, colorectal cancers as well as NSCLCs) that address downstream pathways controlled by KRAS. We discussed examples of these strategies in our book-length report Multitargeted Therapies: Promiscuous Drugs and Combination Therapies, published by Cambridge Healthtech Institute/Insight Pharma Reports in 2011. Strategies discussed in that report are based on the finding that KRAS controls signal transduction via two key pathways: the B-Raf-MEK-ERK pathway and the PI3K-Akt pathway. This is illustrated in the figure at the top of this article. As discussed in our 2011 report, researchers are attempting to develop treatments of mutant-KRAS tumors that involve combination therapies with an inhibitor of the mitogen-activated protein kinase (MEK) together with an inhibitor of phosphatidylinositol 3-kinase (PI3K). Researchers are also attempting to develop combination therapies of MEK inhibitors with standard cytotoxic chemotherapies, which if successful will avoid having to use combinations of two expensive targeted therapies.

In the co-clinical trial that is the focus of the 29 March 2012 Nature research report and News and Views commentary, researchers developed a genetically-engineered mouse model to study treatment of mutant-KRAS NSCLCs with either the antimitotic chemotherapy drug docetaxel alone, or docetaxel in combination with the MEK kinase inhibitor selumetinib (AZD6244, AstraZeneca). In the parallel human clinical trial, researchers are also studying treatment of patients with mutant-KRAS NSCLC with docetaxel alone or docetaxel plus selumetinib. (There is no treatment arm in the human clinical trial in which patients are treated with selumetinib alone, since selumetinib monotherapy of NSCLC patients had shown no efficacy in a previous Phase 2 study; this was confirmed in mouse model studies.)

In humans with mutant-KRAS NSCLC, many tumors with mutations in KRAS have concomitant genetic alterations in other genes that may affect response to therapy. Therefore, the co-clinical trial researchers wished to design mouse models with lung tumors with either Kras mutations alone or with mutations in both Kras and another gene that is often concomitantly mutated in mutant-KRAS NSCLCs in humans. The researchers therefore constructed mouse models with cancers bearing the activating Kras(G12D) mutation, either alone or together with an inactivating mutation in either p53 or Lkb1. The researchers achieved this via a conditional mutation system using nasal instillation of specifically genetically-engineered adenoviruses. As result, a small percentage of lung epithelial cells harbored these mutations. It is from these cells that the NSCLC-like tumors arose, analogous to the clonal origin of sporadic lung tumors in humans.

Of the two tumor suppressor genes that are frequently mutated in human mutant-KRAS NSCLCs and that were modeled by the co-clinical trial researchers, p53, often called the “guardian of the genome”, is familiar to most of you. The other gene, Lkb1 [liver kinase B1, also known as serine/threonine kinase 11 (STK11)], was discussed in an earlier article on the Biopharmconsortium Blog, entitled “The great metformin mystery–genomics, diabetes, and cancer.”

LKB1 (whether in regulation of gluconeogenesis in the liver or in its role as a tumor suppressor) acts by activation of AMPK (AMP-activated kinase, a sensor of intracellular energy status.) In lung cancer (as shown by the same group that performed the 2012 co-clinical trial), LKB1 acts to modulate lung cancer differentiation and metastasis. Germline mutations in LKB1 are associated with the familial disease Peutz-Jegher syndrome, in which patients develop benign polyps in the gastrointestinal tract. Studying a mouse model of mutant-LKB1 Peutz-Jeger syndrome, Reuben J. Shaw (Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA, who was prominently mentioned in our “great metformin mystery” article) and his colleagues showed that the LKB1-AMPK pathway downregulates the mTOR pathway–specifically the rapamycin-sensitive mTOR complex 1 (mTORC1) and its downstream effector hypoxia-inducible factor-1α (HIF-1α). HIF-1α expression in turn upregulates the expression of its downstream effectors hexokinase II and glucose transporter 1 (GLUT1), which are involved in cellular utilization of glucose. LKB1-deficient polyps in this mouse model thus show increased expression of hexokinase II and GLUT1, resulting in dramatically increased glucose utilization.

In the new co-clinical trial, genetically-engineered mice that showed established lung tumors [as determined via magnetic resonance imaging (MRI)] were randomized to receive either docetaxel, selumetinib, or a combination of the two drugs. For tumors with only a Kras mutation, treatment with docetaxel alone resulted in a modest rate of response, with 30% of mice showing a partial response. Mice that bore mutant-Kras tumors that also had mutations in either p53 or Lkb1 had much lower rates of response to docetaxel monotherapy (5% and 0%, respectively), and more of these mice showed progressive disease on MRI or died of their disease. Of mice treated with the docetaxel/selumetinib combination, those with single-mutant Kras tumors showed a 92% overall response rate, and those with mutant Kras/p53 tumors showed a 61% overall response rate. However, mice with mutant Kras/Lkb1 cancers showed only a modest response to the docetaxel/selumetinib combination; 33% of mice achieved a partial response. The difference in response rate of mice with Kras/Lkb1 tumors to docetaxel/selumetinib compared to the other two genotypes was found to be statistically significant.

Using the genetically-engineered NSCLC mouse model in biomarker development

In human patients in clinical trials or in treatment for their cancers, performing repeated biopsies to monitor treatment is difficult. The co-clinical trial researchers therefore wished to develop less invasive means of monitoring both co-clinical and clinical trials of docetaxel/selumetinib treatment of NSCLC. They therefore tested the use of positron emission tomography (PET) with 18F-fluoro-2-deoxyglucose (FDG-PET) as an indicator of early response to therapy that could be used in the clinic. Prior to its radioactive decay (109.8 minute half -life), 18F-FDG is a nonmetabolizable glucose analogue that moves into cells that is preferentially taken up by high-glucose utilizing cells. The researchers found that both Kras/p53 and Kras/Lkb1 tumors showed higher FDG uptake in vivo in the mouse model than did single-mutant Kras tumors. As expected from the earlier study, GLUT1 expression was elevated in Kras/Lkb1 mutant tumors. In human patients, pre-treatment, mutant KRAS/LKB1 tumors also showed a higher FDG uptake that did KRAS tumors negative for LKB1.

Treatment of the mice with docetaxel alone gave no significant changes in FDG uptake in Kras, Kras/p53, or Kras/Lkb1 tumors in vivo. However, within 24 hours of the first dosing of docetaxel/selumetinib, FDG uptake was markedly inhibited in Kras and Kras/p53 tumors. In contrast, treatment of mice with Kras/Lkb1 mutant tumors gave no appreciable decrease in FDG uptake in these tumors. These results show that early changes in tumor metabolism, as assessed by FDG-PET, predict antitumor efficacy of docetaxel/selumetinib treatment.

The FDG-PET study in this mouse model supports the use of this imaging method as a biomarker to monitor the course of treatment in humans.

Cellular signaling in mutant Kras, Kras/p53, and Kras/Lkb1 tumors

The researchers assessed activation of relevant intracellular pathways in tumors in treated and untreated mice with mutant Kras, Kras/p53, and Kras/Lkb1 lung cancers. They performed these studies using two different methods–immunostaining of cancer nodules for phosphorylated ERK, and immunoblotting of tumor lysates. In untreated mice, Kras/Lkb1 tumors show less activation of ERK than do Kras and Kras/p53 tumors, with Kras/p53 tumors showing the greatest amount of activation of the MEK-ERK pathway. Docetaxel had no discernible effect on signaling via the MEK-ERK pathway. Selumetinib alone resulted in decreased ERK activation in Kras and Kras/p53 tumors, but there was still residual activity. The docetaxel/selumetinib combination, however, was more effective in eliminating ERK activation. Pharmacokinetic studies indicated that selumetinib levels were higher in both serum and tumors of mice treated with docetaxel/selumetinib that in those treated with selumetinib alone; this might account for the more potent suppression of MEK-ERK signaling by the combination therapy as compared to selumetinib monotherapy. The researchers studied MEK-ERK activation (as determined by phospho-ERK staining) in a set of 57 human NSCLC tumors with known RAS, p53 and LKB1 mutation status. As with the tumors in the mouse model, of seven patients whose tumors harbored the KRAS activating mutation, the three patients with concurrent p53 mutations showed higher levels of ERK activation.

The decreased activation of ERK in Kras/Lkb1 tumors suggested that these tumors utilize other pathways to drive their proliferation. On the basis of their prior studies of signal transduction in mutant-Lkb1 lung tumors, the researchers focused on AKT and SRC. Immunoblotting studies showed that Kras/Lkb1 mutant tumors had elevated activation of both AKT and SRC. As one can see from the figure at the top of this article, AKT is a downstream effector of PI3K; since the PI3K/AKT pathway regulates expression of GLUT1 and hexokinase, increased activation of the PI3K/AKT pathway is consistent with the increased uptake of FDG of mutant Kras/Lkb1 tumors. In the figure, SRC (which is not shown) represents one of the major “other effectors” controlled by RAS. These results indicate that concomitant mutation of Lkb1 in mutant-Kras NSCLCs may shift the signaling pathways that drive tumor proliferation from MEK-ERK to PI3K/AKT and/or SRC. This shift would result in primarily resistance of Kras/Lkb1 tumors to treatment with docetaxel/selumetinib.

Long-term benefits of treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel/selumetinib as opposed to docetaxel monotherapy

The researchers studied long-term treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel monotherapy versus docetaxel/selumetinib. In mice with mutant-Kras tumors, treatment with docetaxel monotherapy gave stable disease for several weeks, while docetaxel/selumetinib treatment resulted in tumor regression and slower regrowth of tumors. The addition of selumetinib to docetaxel significantly prolonged progression-free survival in these mice. In mice with Kras/p53 tumors, treatment with docetaxel alone resulted in progressive disease, but docetaxel/selumetinib treatment resulted in initial disease regression followed by progression, resulting in prolonged progression-free survival. These results indicate that treatment with combination therapy as opposed to docetaxel alone results in improved progression-free survival, but not cure, in mice with Kras- and Kras/p53-mutant tumors.

The researchers also investigated mechanisms of acquired tumor resistance in mice with mutant-Kras and Kras/p53 tumors, which had been treated long-term with docetaxel/selumetinib. In moribund animals that had received this treatment, all tumor nodules examined showed a recurrence of ERK phosphorylation. This suggested that acquired resistance could be at least in part due to reactivation of MEK–ERK signaling despite ongoing treatment with selumetinib. Evaluation of resistant tumor nodules suggested that more than one mechanism for pathway reactivation was occurring; study of these mechanisms is ongoing.

Conclusions of the new co-clinical study

The results of this co-clinical study predict that docetaxel/selumetinib combination therapy will be more effective than docetaxel monotherapy in several sub-classes of mutant-KRAS NSCLC. This prediction is consistent with the early results of a Phase 2 clinical trial of these two drug combinations in second-line treatment of patients with KRAS-mutant NSCLC.

However, the co-clinical trial also predicts that concurrent mutation of LKB1 in mutant-KRAS tumors will result in primary resistance to docetaxel/selumetinib combination therapy, perhaps via activation of parallel signaling pathways such as AKT and SRC. Since LKB1 status is not being prospectively assessed in the ongoing human clinical trial, the presence of patients with cancers having concurrent LKB1 mutations may diminish the differences between treatment arms based solely on KRAS status. The results of the co-clinical trial suggests that researchers perform retrospective analysis of p53 and LKB1 status in samples from the concurrent human clinical trial. Future clinical trials should then be designed that involve prospective analysis to ensure sufficient enrollment of patients with all three genotypes to enable sufficiently powered sub-group analyses.

Although the results of the co-clinical trial indicate that patients with mutant KRAS/LBK1 tumors be excluded from trials of docetaxel/selumetinib treatment, the research group that has been conducting the co-clinical trial has also been conducting studies that may lead to treatment strategies for KRAS/LBK1 tumors.

The co-clinical trial also allowed researchers to design and validate biomarker strategies, specifically the potential use of the less-invasive FDG-PET to predict efficacy and to monitor treatment. The co-clinical animal-model study also enabled the discovery of mechanisms of both primary and acquired resistance that might benefit future clinical trials and discovery/development of drugs. (The studies on acquired resistance are in a early stage and are ongoing). Any mechanisms of acquired resistance discovered in co-clinical studies should be confirmed in human clinical trials by examining biopsy samples from patients who relapse on therapy. The ability to assess mechanisms of resistance in preclinical or co-clinical animal studies may enable researchers to design rational drug combination strategies that can be implemented in future clinical studies.

The results of the new co-clinical trial strengthens the contention that co-clinical trials in genetically-engineered mice can provide data that can predict the outcome of parallel human clinical trials. Co-clinical trials can also be used to generate new hypotheses for use in analyzing concurrent human trials, and for design of future clinical studies. Moreover, co-clinical trials can result in the validation of improved animal models for human cancers, which can be used in research and preclinical testing of oncology agents, and in validation of biomarkers for clinical studies in oncology. Given the inadequacy of “standard” xenograft models, which is a major factor in the high attrition rate of pipeline oncology drugs, the availability of validated genetically-engineered animal models may be expected to enable improved oncology drug development.

__________________________________________

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.

Posted in Animal Models, Biomarkers, Cancer, Drug development, Metabolism, Personalized medicine, Strategy and Consulting, Translational medicine

How can we fix the clinical trial system?

Published by Allan Haberman on October 28, 2011 | Leave a response

http://bit.ly/dGrWW3

In recent months, there have been quite a few articles on the need to fix the clinical trial system. Among the most recent articles is the one by Boston-based Nature writer Heidi Ledford, Ph.D. published as a News Feature in the 29 September issue of Nature. In my humble opinion, this is the best article on the subject among those that have been published recently.

The pharmaceutical/biotechnology industry is frustrated with the increasing expense and the low output of the clinical trial system. This low productivity is economically unsustainable. The current clinical trial paradigm is over 50 years old. Back in the 1960s, the norm was to conduct single trials at single sites, each designed to answer a single question.

Nowadays, the norm is the large, multicenter clinical trial, especially for Phase 3 trials. “Multicenter” means that a trial is conducted at multiple sites, often in different countries, and could involve thousands of investigators and staff members. As pointed out in Dr.Ledford’s article, the large trials are mandated by the need in our more risk-adverse world to detect safety issues that occur in only a small percentage of patients, and to obtain good statistics for drugs that confer only a small benefit over the standard of care. However, certain major diseases require large trials of long duration even for drugs that may confer large benefits. For example, because the percentage of patients per year in cardiovascular disease (CVD) trials who experience cardiovascular events is small, these trials must be large and multiyear, in order to see any benefit even for a breakthrough drug.

The advent of personalized medicine–developing drugs and combinations of drugs that are specific for the molecular mechanism behind a patient’s disease–has put additional burdens on the clinical trial system. A disease may be found to be a collection of rare diseases in terms of mechanistic subtypes, each of which affects only a small number of people. This makes patient recruitment difficult.

As stated by Dr.Ledford, “Solving the problem may require fundamental changes to the clinical-trial system to make it faster, cheaper, more adaptable and more in tune with modern molecular medicine.”

Don’t use an “e-commerce” approach to determining drug efficacy!

Other commentators have recently noted the need to make clinical trials “faster, cheaper, and more adaptable.” Several of them have suggested bringing in strategies from other industries, especially e-commerce and social media.

For example, in an editorial published in the 23 September issue of Science, Andrew Grove, the former Chief Executive Officer of Intel, proposes moving towards an “e-trial” system, based on such large-scale e-commerce platforms as that of Amazon.com. Under the proposed e-trial system, the FDA would ensure safety only, not efficacy, and would continue to regulate Phase 1 trials. Once Phase 1 trials have been successfully completed, patients would be able to obtain a new drug through qualified physicians.

Patients’ responses to a drug would be stored in a database, along with their medical histories. There would be measures to protect a patient’s identity, and the database would be accessible to qualified medical researchers as a “commons.” The response of any patient or group of patients to a drug or treatment could then be tracked and compared to those of others in the database who were treated in a different manner or were untreated. These comparisons would provide insights into a drug’s efficacy, and how individuals or subgroups (perhaps defined in part via biomarkers) respond to the drug. This would liberate clinical trials from the “tyranny of the average” that characterize most trials today. As the database grows over time, analysis of the data would also provide information needed for postmarketing studies and comparative effectiveness studies.

Dr. Grove’s proposal is one of several in which the mandate of the FDA (and regulatory agencies in Europe, Japan, etc.) is to regulate safety only (via Phase 1 clinical trials) not efficacy. Efficacy is then determined via some sort of open system, with information gathered and provided to patients and physicians electronically, via systems reminiscent of e-commerce or social media.

We are opposed to removing efficacy from the oversight of the FDA and other regulatory agencies. There are two reasons for this, both of which are illustrated graphically in Box 1 of Dr. Ledford’s article, entitled “the clinical trial cliff”. Approximately half of Phase 2 clinical trials between 2008 and 2010 failed due to inability to demonstrate efficacy. (Around one-third of Phase 2 failures were due to safety, and the remaining failures were mainly due to strategic decisions to terminate a drug.) Among Phase 3 failures between 2007 and 2010, around two-thirds were due to efficacy, and around one-quarter were due to safety. These results indicate that the majority of drugs entered into clinical trials lack efficacy.

The second reason is that many safety problems–especially the rarer safety issues that occur in only a small percentage of patients–are typically not detected in Phase 1, but in Phase 3 and even the postmarking period.

Reduce clinical attrition with new trial designs and improved animal models

Dr. Ledford’s proposals for fixing clinical trials leave regulatory agencies in charge of overseeing both safety and efficacy. They mainly focus on improving clinical trials by reducing “attrition”–i.e., failure of drugs in the clinic, especially in Phase 2 and Phase 3, and on improving patient recruitment. Haberman Associates has produced publications–as well as articles on this blog–during the 2009-2011 period that provide a more in-depth discussion of strategies for reducing attrition than is possible in a 3-page article such as Dr. Ledford’s.

Two of Dr. Ledford’s strategies involve modifications of clinical trial design. Both of these are discussed in Chapter 6 of our book-length Cambridge Healthtech Institute (CHI) Insight Pharma Report, Approaches to Reducing Phase II Attrition. The first is the “Phase 0″ trial. This is a type of pre-Phase 1 clinical trial, which uses microdoses of a drug to assess such parameters as pharmacokinetics and target occupancy. As Dr. Ledford suggests, in some cases Phase 0 trials can reduce or eliminate pharmacological testing in animals, and allow researchers to get human data more quickly.

The other trial design strategy mentioned in Dr, Ledford’s article is the use of adaptive clinical trials. This type of trial allows researchers to change the course of a trial in response to trial results. For example, this may mean assigning new patients to specific doses, changing the numbers of patients assigned to each arm of a trial, and changes in hypotheses or endpoints. Monitoring and changing the trial is typically done by an independent data monitoring committee [DMC] so that ideally, double-blind conditions are maintained.

As Dr. Ledford states, adaptive clinical trials may result in shortening the time and cost of the clinical trial process. But, as with Phase 0 microdosing trials, there are many controversies surrounding adaptive clinical trials. Both of these strategies are works in progress.

The other strategy for reducing attrition discussed in Dr. Ledford’s article is to use improved animal models (i.e., animal models designed to more faithfully model human disease) in preclinical studies. We discussed this strategy in Approaches to Reducing Phase II Attrition, and in greater detail in another book-length report, Animal Models for Therapeutic Strategies. I also recently led the workshop “Developing Improved Animal Models in Oncology and CNS Diseases to Increase Drug Discovery and Development Capabilities” at Hanson Wade’s 2011 World Drug Targets Summit.

Several articles on our Biopharmconsortium Blog also focus on improved animal models for predicting efficacy of drug candidates in discovery research and in preclinical studies. Our April 15, 2010 blog post, based on an article in The Scientist, focused on “co-clinical mouse/human trials”. This type of clinical trial was developed by Pier Paolo Pandolfi, MD, PhD (Director, Cancer and Genetics Program, Beth Israel-Deaconess Medical Center Cancer Center and the Dana-Farber/Harvard Cancer Center) and his colleagues.

These trials utilize genetically engineered transgenic mouse strains that have genetic changes that mimic those found in specific human cancers. These mouse models spontaneous develop cancers that resemble the corresponding human cancers. In the co-clinical mouse/human trials, researchers simultaneous treat a genetically engineered mouse model and patients with tumors that exhibit the same set of genetic changes with the same experimental targeted drugs. The goal is to determine to what extent the mouse models are predictive of patient response to therapeutic agents, and of tumor progression and survival. The studies may thus result in validated mouse models that are more predictive of drug efficacy than the currently standard xenograft models.

The new Ledford Nature article discusses co-clinical trials as a means to develop more predictive animal model studies–not only using improved, potentially more predictive animal models, but also treating these animals in similar way (in terms of doses, formulations, schedules of medication, etc.) to the humans in the parallel human clinical trial.

The Ledford article mentions the animal-model portion of a co-clinical trial, which was published in January 2011. This trial utilized two genetically-engineered PDGF (platelet-derived growth factor)-driven mouse models of the brain tumor glioblastoma multiforme (GBM), one of which has an intact PTEN gene and the other of which is PTEN deficient.

Unlike the “standard” mouse xenograft models, these models more closely mimicked the human disease, including growth of tumors within the brain, not subcutaneously. Thus any drug administered to these mice systemically (e.g., intraperitoneally, as was done in this study) had to cross the blood-brain barrier (BBB), as in the case of human clinical trials. This would not be the case with a standard xenograft model, which is one deficiency of these models for brain tumors such as GBM.

GBM is both the most common and the most malignant primary brain tumor in adults. It has a poor prognosis. PDGF-driven GBMs, which results from deregulation of the PDGF receptor (PDGFR) or overexpression of PDGF, account for about 25-30% of human GBMs. These mutations result in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. These tumors may also exhibit mutation or loss of heterozygosity of the tumor suppressor PTEN, which also upregulates the PI3K/Akt/mTOR pathway.

The researchers tested the Akt inhibitor perifosine (Keryx Biopharmaceuticals, an alkylphospholipid) and the mTOR inhibitor CCI-779 (temsirolimus; Pfizer’s Torisel; originally developed by Wyeth prior to the Pfizer merger and approved for treatment of renal cell carcinoma), both alone and in combination, in vitro and in vivo. Specifically, the drugs and drug combinations were tested in cultured primary glioma cell cultures derived from the PTEN-null and PTEN-intact mouse PDGF-driven GBM models, and in the animal models themselves.

The studies showed that both in vitro and in vivo, the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null cells or animals was achieved by using both inhibitors in combination. In vivo, the decreased Akt and mTOR signaling seen in mice treated with the combination therapy correlated with decreased tumor cell proliferation and increased cell death; these changes were independent of PTEN status. The co-clinical animal study also suggested new ways of screening GBM patients for inclusion in clinical trials of treatment with perifosine and/or CCI-779.

According to Dr. Ledford’s Nature article, the National Cancer Institute (NCI) invested $4.2 million in Dr. Pandolfi’s co-clinical trials in prostate and lung cancer in 2009. In addition to the co-clinical trials with genetically-engineered mouse models run by Dr. Pandolfi and others, researchers at the Jackson Laboratory are conducting co-clinical trials with mouse xenograft models that receive tumor cells from patients to be treated in human clinical trials.

Use patient registries in recruitment of patients for clinical trials

In Dr, Ledford’s article, she discusses a crucial factor other than clinical attrition that hinders progress in conducting clinical trials–patient recruitment. According to the article, at least 90% of trials are extended by at least six weeks because of failure to enroll patients on schedule. Only about one-third of the sites involved in a typical multicenter trial manage to enroll the expected number of patients. As a result, clinical trials are longer and more expensive, and some of them are never completed.

Personalized medicine, in which researchers use biomarkers or other criteria to determine what fraction of patients with a particular disease are eligible for a trial (e.g., cancer patients with an activating mutation in a kinase that is the target of the drug to be tested), makes recruitment harder. That is because researchers must screen large numbers of patients to identify the fraction of patients that would be eligible for the trial. So they need to recruit (and screen) a much larger number of patients than in conventional clinical trials with no patient stratification.

Therefore, researchers, “disease organizations”, and patient advocates are devising new strategies to facilitate recruitment of eligible volunteers. Dr. Ledford cites the example of the Alpha-1 Foundation (Miami, Florida), a “disease organization” that focuses on the familial disease alpha-1 antitrypsin deficiency. (This disease renders patients susceptible to lung and liver diseases.) This foundation has created a registry of patients with alpha-1 antitrypsin deficiency who are willing to be contacted about and to participate in clinical trials.

There are also cancer registries. Dr. Ledford mentions the Total Cancer Care program run by the Moffitt Cancer Center (Tampa, Florida). This program, which involves 18 hospitals, compiles medical history, tissue samples (stored for future analysis) and genetic information about each patient’s tumor. Patients can consent to doctors contacting them about trials. There are other similar programs being developed in the Netherlands and elsewhere. Dr.Ledford mentions the difficulty in negotiating agreements between institutions, and the need for adequate, ultra-secure networks to support registries that connect multiple hospitals and research centers.

Patient registries that are designed to proactively support recruitment for clinical trials have some resemblance to a “social media” approach to recruitment. However, there is a big difference–the need to secure the privacy of patient records. The current trend in social media (and in some e-commerce platforms) is anti-privacy. This is yet another important reason why a social media or e-commerce approach to clinical trials or other aspects of biotech/pharma R&D is not a suitable model. (To his credit, Dr. Grove mentions the need to maintain patient privacy and confidentiality. But this is not the norm with e-commerce and social media.)

Cutting red tape for faster and cheaper clinical trials

Dr Ledford also mentions ways to deal with more bureaucratic issues that can slow down or block the progress of clinical trials. The NCI is now initiating a data-management system that will standardize data entry across all 2,000 sites that conduct NCI-sponsored trials. This should help reduce costs and cut down on record-keeping errors and omissions.The FDA is also looking into ways to reduce reporting requirements and paperwork. so that investigators can submit summaries of case reports rather than each individual document.

To adapt to the multicenter nature of clinical trials, the US Office for Human Research Protections (Rockville, Maryland), which oversees NIH-funded human studies, has proposed changes to its guidelines that would require designation of a single review board for each project. This may greatly improve the current situation, in which multicenter trials must get approval from each center’s institutional review board. This can take months or even years. Despite the definite advantages of more centralized review, individual research centers may be reluctant to give up their direct oversight of clinical trials.

Something important was not in Dr. Ledford’s article

The space limitations for Dr. Ledford’s “News Feature” article, plus its strict focus on clinical trials per se, did not permit her to include something of crucial importance to reduce clinical attrition. That is utilizing such strategies as biology-driven drug discovery in the research phase of drug development. These strategies are designed to select the best targets and to discover drugs that are more likely to be efficacious in treating a particular group of patients. These research strategies are then coupled with early development strategies that emphasize designing clinical trials aimed at obtaining rapid proof of concept in humans. Such trials typically involve the use (and often the discovery) of biomarkers.

We discussed these issues extensively in our report, Approaches to Reducing Phase II Attrition, as well as in an article published in Genetic Engineering and Biotechnology News (and available on our website) “Overcoming Phase II Attrition Problem“. We also discussed a specific case of the use of this strategy in our October 25, 2010 article on this blog.

Conclusions

Given the low productivity of pharmaceutical R&D, it is tempting to take an envious look at the success of e-commerce and social media, and to attempt to devise strategies that apply methodologies from these industry sectors to the biotech/pharmaceutical industry. We should remember, however, that not so long ago some pharmaceutical executives attempted to apply methodologies from such industries as aerospace, computer hardware, and the auto industry to pharma R&D. Not only did that not work too well for the pharmaceutical industry, but as we all know, the industries that served as a model for these approaches haven’t done very well in recent years either.

In contrast, pharmaceutical and biotechnology companies that have formulated strategies that embrace the uniqueness of biology, such as Novartis and Genentech (the latter now merged with Roche), have done a lot better.

There are other strategies for making clinical trials faster, cheaper, and better that are now under discussion in the biotech/pharma industry and the FDA. These strategies are based on clinical experience, not e-commerce. We shall discuss them in further blog posts.

__________________________________________

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.

Posted in Animal Models, Biomarkers, Cancer, Cardiovascular disease, Drug development, Drug discovery, Haberman Associates, Personalized medicine, Strategy and Consulting, Translational medicine

World Drug Targets Summit, Cambridge MA, July 19-21

Published by Allan Haberman on July 27, 2011 | Leave a response

Hanson Wade’s World Drug Targets Summit took place on July 20-21, 2011, with pre-conference workshops on July 19. The conference was held in the Sheraton Commander Hotel in Harvard Square in Cambridge, MA.

I led the first workshop on the 19th, on “Developing Improved Animal Models in Oncology and CNS Diseases to Increase Drug Discovery and Development Capabilities”. The workshop was well-attended, with good questions and discussion from those in attendance. For a description of the workshop, see our July 5, 2011 blog post. The second workshop, on “Exploiting Kinase Signaling Pathways: Opportunities for Drug Development”, was led by Kamal D Puri and Heather Webb, both of Gilead Sciences (Foster City, CA).

The main conference included speakers from both Big Pharmas (Novartis, UCB Pharma, Merck, Pfizer, AstraZeneca, Boehringer Ingelheim, Bayer Schering Pharma) and such biotech companies as Gilead, Infinity Pharmaceuticals, Merrimack Pharmaceuticals, NeurAxon, and FORMA Therapeutics, as well as a couple of researchers from Harvard Medical School and its teaching hospitals. Attendees who were not speakers included people from these same companies and from other Big Pharmas, as well as from such up-and-coming biotechs as Aileron Therapeutics and Proteostasis Therapeutics (both in Cambridge, MA and both mentioned on our blog), and other companies in the U.S. and in Europe.

In addition to case studies and strategies for identifying and validating drug targets that would be likely to yield safe, efficacious, and commercializable drugs, there was a section on strategies for fostering outsourcing and collaboration in target identification and validation. These included Bayer’s Grants 4 Targets program and Tempero Pharmaceuticals’ collaborative programs. (Tempero is a wholly owned subsidiary of GlaxoSmithKline located in Cambridge, MA.)

One highlight of the Summit was a section on “undruggable” targets (and hard targets known as “high-hanging fruit”); this section occurred at the end of the conference. John Andrews of NeurAxon (Mississauga, Ontario Canada) gave an overview of companies working on “undruggables”, which included not only protein-protein interactions (PPIs), but also what we have called areas of “premature technology” such as RNAi therapeutics and, up until the mid-1990s, monoclonal antibody drugs. (See our blog articles located here, here, and here.) He then presented NeurAxon’s own work on developing a first-in-class neuronal nitric oxide synthase (nNOS) inhibitor for treatment of migraine. nNOS inhibitors represent “high-hanging fruit” because of the difficulty of designing drug-like compounds that are selective for nNOS as opposed to endothelial NOS (eNOS).

At the end of Dr. Andrews’ presentation, I briefly outlined the concept of “premature technologies”, and the development of enabling technologies to overcome technological prematurity. MAb drugs constitute a classic case. I then asked if researchers were developing enabling technologies to make possible the efficient discovery of small-molecule drugs to address PPIs, as opposed to the case-by-case development of such drugs as occurs now. (See this article on our blog for an example.)

The chairman for the day, David Winkler of Infinity Pharmaceuticals, instead of having Dr. Andrews answer the question, moved on to the final speaker of the day, Mark Tebbe of FORMA Therapeutics (Cambridge, MA). Dr. Tebbe discussed FORMA’s technology platforms, which are designed to be enabling technologies for discovery of small-molecule drugs to address PPIs, thus answering my question.

In particular, Dr. Tebbe cited FORMA’s CS-Mapping platform, which enables company researchers to interrogate PPIs in intracellular environments, to define hot spots on the protein surfaces that might constitute targets for small-molecule drugs. (For an example of hot spots that are critical for binding in a PPI in the Wnt signaling pathway, see this research report, which we cited in our PPI blog article.) FORMA combines CS-Mapping technology with its chemistry technologies (e.g., structure guided drug discovery, diversity orientated synthesis) to discover drugs.

As an example of hot spot determination, Dr. Tebbe cited the GTP/GDP biding site of the RAS protein. RAS is a notoriously “undruggable” target that is important in a large percentage of human cancers. As discussed on the company’s website, FORMA has a collaboration with the Leukemia & Lymphoma Society to discover and develop small-molecule compounds that target the interaction between the transcriptional repressor Bcl-6 and the SMRT co-repressor. This interaction is key to signaling pathways that are involved in diffuse large B cell lymphoma, a type of aggressive non-Hodgkin’s lymphoma.

FORMA has several executives and board members with Novartis backgrounds, and Novartis is an investor in FORMA and collaborates with FORMA in the area of small-molecule drugs for PPIs in oncology. As discussed in the blog article mentioned earlier on development of small-molecule drugs to target PPIs, Novartis has also been collaborating with researchers at Harvard teaching hospitals in that area. These collaborations show the interest of Novartis in the PPI area, which many pharmaceutical companies shun because of its difficulty and high risk.

The World Drug Targets Summit was a relatively small conference, but had a high concentration of pharmaceutical and biotechnology company R&D leaders, especially in target identification and validation. This provided excellent opportunities to ask questions of the speakers, and to interact with speakers and other attendees during breaks, and in the “speed networking” session and at the conference’s networking dinner. All and all, it was a good conference.

Posted in Animal Models, Cancer, Chemistry, Drug development, Drug discovery, Haberman Associates, Monoclonal Antibodies, Strategy and Consulting

Update: Workshop on improved animal models for pharma R&D at the World Drug Targets Summit, July 2011

Published by Allan Haberman on July 5, 2011 | Leave a response

The time for the July 2011 World Drug Targets Summit in Cambridge MA is looming closer and closer! Registration for the conference is still open, however.

I will lead a workshop entitled “Developing Improved Animal Models in Oncology and CNS Diseases to Increase Drug Discovery and Development Capabilities” at the Summit on July 19. A workshop on addressing kinase signaling in drug discovery and development will take place later that day. The main conference follows on July 20-21. I am planning to attend the entire conference.

Our workshop will be a discussion of four 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.

There will be one case study on a zebrafish cancer model, two on mouse cancer models, and one on a mouse CNS disease model. The case studies will include applications of these animal models to understanding disease biology, developing new therapeutic strategies, overcoming resistance to breakthrough targeted cancer therapeutics, and identifying drug candidates and advancing them into the clinic.

The main conference will focus on developing improved target discovery and validation strategies that are capable of meeting the challenges of drug discovery and development in the early 21st century–minimizing drug attrition in the clinic, and delivering commercially differentiated products that address unmet medical needs to the market. Speakers will include target discovery and validation leaders from leading pharmaceutical companies, biotechnology companies, and academic institutions.

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

Posted in Animal Models, Cancer, Drug development, Drug discovery, Haberman Associates, Psychiatric diseases, Strategy and Consulting, Translational medicine

Workshop on improved animal models for pharma R&D at the World Drug Targets Summit, July 2011

Published by Allan Haberman on May 12, 2011 | Leave a response

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.

Posted in Animal Models, Cancer, Drug development, Drug discovery, Haberman Associates, Neurodegenerative diseases, Strategy and Consulting

FDA approves ipilimumab (Medarex/Bristol-Myers Squibb’s Yervoy) for treatment of metastatic melanoma

Published by Allan Haberman on March 30, 2011 | Leave a response

Melanoma

On March 25, 2011, the FDA approved ipilimumab (Medarex/Bristol-Myers Squibb’s [BMS's] Yervoy) for treatment of unresectable or metastatic melanoma. The drug has been approved for patients with either newly-diagnosed or previously-treated disease.

According to Richard Pazdur, the director of the FDA’s office of oncology drug products, none of the previously-approved treatments for metastatic melanoma, a disease with a poor prognosis, prolonged a patient’s life. “Yervoy is the first therapy approved by the FDA to clearly demonstrate that patients with metastatic melanoma live longer by taking this treatment.”

We discussed ipilimumab briefly in a previous article on this blog. As we stated in that article, the results of a Phase 3 trial of ipilimumab were published in the August 19, 2010 issue of the New England Journal of Medicine. Ipilimumab is an immunomodulator that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) to potentate an antitumor T-cell response. The drug is a monoclonal antibody (MAb). In this NEJM article, the researchers reported that ipilimumab treatment–given with or without a gp100 peptide vaccine–showed a median overall survival of 10 months, as compared to 6.4 months in patients receiving gp100 alone. Ipilimumab treatment also gave improved one-year survival compared with gp100 alone–46% versus 25%. Two-year survival was 24% in the ipilimumab group and 14 percent in the gp100 group.

Decision Resources published our report on development of immunomodulators in treatment of cancer in 2007. This report includes a discussion of ipilimumab, and provides further information on its mechanism of action, adverse effects, etc., as well as on other immunomodualtors for treatment of cancer, some of which are now on the market.

BMS plans to report on the results of a later Phase 3 study, which also demonstrated significantly improved survival as compared to a control treatment, at the American Society of Clinical Oncology (ASCO) meeting in Chicago in June.

In its March 25, 2011 press release, BMS said that it had agreed with the FDA to conduct a post-marketing study comparing the safety and efficacy of the 3 mg/kg dose vs. an investigational 10 mg/kg dose in patients with unresectable or metastatic melanoma.

The Full Prescribing Information for ipilimumab will include a boxed warning for immune-mediated adverse effects. Ipilimumab treatment can result in severe or fatal immune-mediated adverse effects, especially enterocolitis, hepatitis, dermatitis, neuropathy, or endocrinopathy. These are usually reversible by discontinuing ipilimumab therapy and treatment with high-dose steroids. According to the FDA, severe to fatal autoimmune reactions were seen in 12.9% of patients treated with the drug.

As part of the approval of ipilimumab, BMS is collaborating with the FDA to develop a Risk Evaluation and Mitigation Strategy, to help inform patients and providers about these safety risks. The company has put in place a system that will enable it to deliver these educational materials to healthcare professionals at the time they order the drug.

Strategic implications for BMS

BMS has hailed the approval of ipilimumab as a victory for its strategic changes over the past several years. The company has been focusing on its pharmaceutical business, selling off such nonpharmaceutical assets as the Mead Johnson Nutrition Company (MJN), and instituting other cost-cutting measures. BMS has at the same time been developing its “String of Pearls” strategy. In this strategy, BMS has been forming a series of acquisitions, alliances and partnerships with biopharmaceutical companies, involving both small molecules and biologics. According to BMS, the String of Pearls strategy has enabled BMS to expand its pipeline by nearly 40 percent. About one-third of BMS’ pipeline drugs are now biologics.

We have discussed the String of Pearls strategy, and two acquisitions that have been part of it, on this blog. These were the acquisition of Medarex (the largest of the “pearls”), and the newest acquisition, ZymoGenetics. It was MAb-therapeutic leader Medarex, now a wholly-owned subsidary of BMS, that initially developed ipilimumab.

BMS faces the expiration of patent protection for its best-selling product, the anticlotting drug Plavix, in 2012. The introduction of ipilimumab, which several analysts expect to become a blockbuster, should help mitigate the results of the Plavix patient expiration. However, ipilimumab is not likely to fully replace the lost sales due to generic competition with Plavix. Moreover, the approval of one drug–ipilimumab–does not necessarily mean that BMS’ new R&D strategy, based on the String of Pearls acquisitions and partnerships, will yield a rich series of important approved drugs in the next 5-10 years. However, ipilimumab itself is such an important drug, in terms of its path-breaking mechanism of action, its addressing unmet medical need in a fatal disease, and its likely blockbuster status.

Another melanoma drug is on the way

The Biopharmconsortium Blog has been following the development of Daichi Sankyo/Plexxikon/Roche’s PLX4032/RG7204 (now designated as vemurafenib) for about a year. We have published several articles on the drug and on related scientific, clinical trial strategy, and business issues. This targeted kinase inhibitor, which is exquisitely specific for the melanoma driver mutation B-Raf(V600E), has been in Phase 3 clinical trials, and its developers filed for U.S. and European approval in May 2011. The drug is expected to reach the market in 2012. As with ipilimumab, Plexxikon and Roche reported that a Phase 3 trial of PLX4032 gave enhanced overall survival as compared with treatment with the standard of care, dacarbazine. The companies also plan to present the results of this trial at the ASCO meeting in June.

Metastatic melanoma patients, who have had few options for treatment, will now have two new, breakthrough drugs that can give them additional months of life, and in some cases longer. However, no treatment now on the horizon will result in long-term survival. In the case of PLX4032, this is due to the development of resistance to the drug. As we discussed previously, researchers are studying mechanisms of PLX4032 resistance, and developing potential combination therapies to overcome it. A clinical trial of at least one combination therapy, in collaboration with Genentech, is planned to begin soon.

A new approach to PLX4032-based combination therapy for melanoma

Meanwhile, another approach to development of an effective combination therapy with PLX4032 comes from an unexpected source.

We had discussed a zebrafish model of melanoma, developed by Leonard Zon’s laboratory at Children’s Hospital/Howard Hughes Medical Institute/Harvard Medical School (Boston, MA), in our 2010 Insight Pharma Report Animal Models for Therapeutic Strategies. In this model, the researchers created transgenic zebrafish strains in which B-Raf(V600E) is expressed under control of the melanocyte-specific mitfa promoter. Wild-type zebrafish expressing B-Raf(V600E) in their melanocytes developed benign nevi, while those with germline mutations in p53 may develop either nevi or melanomas. This suggests these two mutations are necessary, but not sufficient, to cause melanoma. (In humans, nevi may express B-Raf(V600E), which also indicates that it is not sufficient to cause melanoma. And in human melanomas, p53 is either mutated or otherwise rendered inactive.)

Now, in the 24 March issue of Nature, Dr. Zon and his colleagues used this model to study the mechanism of tumorigenesis in melanoma. They found that early-stage embryos of the transgenic zebrafish showed abnormal expansion of neural crest progenitors, and that these progenitors failed to terminally differentiate. (Melanocytes are one of the cell types that develop from the neural crest lineage.) In adult transgenic zebrafish, melanomas develop and are positive for neural crest progenitor markers, and thus appear to retain a neural crest progenitor-like phenotype.

The researchers therefore screened 2,000 compounds to identify those that act as suppressors of neural crest progenitors, without displaying toxicity. The one compound that satisfied these criteria, NSC210627, was similar to brequinar, an inhibitor of dihydroorotate dehydrogenase (DHODH), and NSC210627 also inhibited DHODH in vitro. The researchers therefore tested another more readily-available DHODH inhibitor, leflunomide (Sanofi-Aventis’ Arava). It had the same effects on the zebrafish as NSC210627 and was used for further studies.

Leflunomide treatment resulted in a nearly complete inhibition of neural crest development in zebrafish embryos, and specifically resulted in abrogation of melanocyte development both in zebrafish embryos and in Xenopus (African clawed frog) embryos. The drug’s target, DHODH, catalyzes a step in the synthesis of pyrimidine nucleotides, and thus inhibits transcriptional elongation. The researchers found that leflunomide caused specific defects in the transcriptional elongation of genes necessity for neural crest development in zebrafish. In human melanoma cell lines, leflunomide also inhibited transcriptional elongation in genes necessary for neural crest development and for melanoma growth (e.g, the Myc oncogene, which is required for both processes). Leflunomide (or its active metabolite, A771726) caused inhibition of growth both of human melanoma cell lines in vitro and in vivo in mouse xenograft models, but had little effect on non-melanoma cell lines in vitro. Combined treatment with leflunomide and PLX4032 showed even greater inhibition of growth of human melanoma cells in vitro and in vivo than treatment with either single agent.

Leflunomide is a marketed drug that is approved for treatment of moderate to severe rheumatoid arthritis and psoriatic arthritis. In these diseases, it appears to work via inhibiting the expansion of autoimmune lymphocytes by inhibiting transcriptional elongation in specific genes in these cells. Although leflunomide can have serious adverse effects in a minority of patients (e.g., liver damage), it has a generally favorable safety profile. Dr. Zon and his colleagues suggested that combination therapy of patients whose tumors are positive for B-Raf(V600E) with PLX4032 and leflunomide would be more effective than treatment with either drug alone, and that this combination therapy might help to overcome PLX4032 resistance.

Since leflunomide is already approved by the FDA, and both leflunomide and PLX4032 have been proven to be safe in clinical trials, researchers should be able to readily initiate clinical trials of the combination therapy. Dr. Zon says that he is now working toward initiation of a clinical trial of the drug combination.

________________________________

Posted in Animal Models, Cancer, Drug development, Drug discovery, Strategy and Consulting

2010: Breakthroughs, Newsmakers, and Deals of the Year

Published by Allan Haberman on December 29, 2010 | Leave a response

2010

Breakthroughs of the Year

As it does every year, Science published its “Breakthrough of the Year” for 2010 in the 17 December issue of the journal.

For its “Breakthrough of the Year”, Science chose a non-life science innovation, the first quantum machine. Interestingly, the same issue of Science included a Perspective on biophysicist Britton Chance, who died last November at the age of 97. Among his many accomplishments, Dr. Chance discovered that biological electron transfer operates via quantum tunneling, a mechanism central to photosynthesis, respiration, and many oxidoreductase enzymes. Mitochondria, chloroplasts, and oxidoreductase enzymes thus constitute biological quantum machines of a sort.

Interestingly, Dr. Chance continued to work on metabolism all of his long working life, including in the era of molecular biology when interest in that field waned. By doing so, he made many important contributions, including the mechanism for the generation of the reactive oxygen species (ROS) superoxide and peroxide during normal mitochondrial respiration, and the use of near-infrared (NIR) light for noninvasive diagnostics.

Although Science chose a non-life science advancement as its “Breakthrough of the Year”, the journal’s runners-up for “Breakthrough of the Year” were replete with life science items. The first runner-up was the synthetic Mycoplasma mycoides genome constructed by the J. Craig Venter Institute, which they used to create “the first synthetic cell”. As we discussed in a series of two articles on this blog (see here and here, although the creation of the synthetic genome and the “synthetic cell” represented a technical tour de force, it did not represent a true breakthrough. Many leading scientists, including leaders in the field of synthetic biology, agreed with us. However, at least several bioethicists and philosophers hailed this work as a milestone, calling it “the end of vitalism”. (As we noted in another blog post, however, not all bioethicists agree.)

Moreover, policy-makers were sufficiently alarmed by the “synthetic cell” that (as noted in the Science “Breakthrough of the Year” runners-up article) the Presidential Commission for the Study of Bioethical Issues held hearings on policy implications of this research. Nevertheless, the report of this commission (issued in December 2010) concluded that the Venter research “does not amount to creating life as either a scientific or a moral matter” and that synthetic biology remains “in the early stages,” with any dangers well into the future. The commission recommended continuing White House oversight, but a relatively mild set of regulatory measures.

As we said in our second article on the “synthetic cell”, we are much more impressed by the metabolic engineering studies of Jay Keasling, and by George Church’s automated method for optimizing metabolic engineering pathways, which we had discussed in an earlier blog post. The Science “Breakthrough of the Year” runners-up article mentioned Dr. Church’s automated system, among other synthetic biology advances made in 2009 and 2010.

Meanwhile, in a review of metabolic engineering published in the 3 December 2010 issue of Science, Dr. Keasling says that although minimal bacterial hosts such as Dr. Venter’s “synthetic” mycoplasma may be of scientific interest, they are not suitable to use in metabolic engineering studies whose goal is scale-up for industrial production of medicines, chemicals, or biofuels. This agrees with our statement that such applications require “workhorse” organisms that can take the extensive genetic manipulation needed to engineer new metabolic pathways, and which are capable of scale-up.

We therefore believe that the “synthetic cell” is not the life science breakthrough of the year, despite its placement at the top of Science‘s “Breakthrough of the Year” runners-up article.

Our nominee for the life science breakthrough of the year is listed right under the “synthetic cell” in the Science “Breakthrough of the Year” runners-up article. It is the determination of the sequence of approximately two-thirds of the Neanderthal genome by Svante Pääbo (Max-Planck Institute for Evolutionary Anthropology, Leipzig, Germany.) and his colleagues. This achievement is something that only a few years ago seemed completely impossible. Moreover, this work is of great cultural significance, since it indicates that Neanderthals contributed some 1-4 percent of the genome sequences of non-African present-day humans. More recently, Dr. Pääbo and his colleagues followed up the Neanderthal studies by using their DNA synthesis methods to identify a third species of humans, known as Denisovans. Denisovans, who were more closely related to Neanderthals than to modern humans, were alive at the same time as modern humans emerged from Africa and also encountered the Neanderthals. Dr. Pääbo’s new studies indicate that the Denisovans contributed some 4–6% of the genome sequences of present-day Melanesians.

Despite the importance of the Pääbo Neanderthal studies, we have not blogged on this work simply because it has nothing to do with drug discovery and development. However, perhaps someday, for example, some of the products of genes that are found in present-day humans but not in Neanderthals could emerge as potential drug targets. As discussed in the Science “Breakthrough of the Year” runners-up article, researchers have begun studying some of these gene products in cell culture systems.

Moreover, the types of advanced, next-generation DNA sequencing methods used by Dr. Pääbo and his colleagues are being applied to studies that are relevant to drug discovery. These include the 1000 Genomes Project, which seeks to find all single-nucleotide polymorphisms (SNPs) present in at least 1% of humans. This and other next-generation genomics projects were listed in the Science “Breakthrough of the Year” runners-up article, as the third runner-up. The 1000 Genomes Project, as well as genome-wide association studies (GWAS) that use high-throughput DNA sequencing methods, may enable researchers to identify rare mutations that are involved in complex human diseases. This might in turn lead to the discovery of novel drugs and diagnostics.

Among other life science items in the Science “Breakthrough of the Year” runners-up article was the production of knockout rats. We discussed knockout rats in an October 1, 2010 blog post.

Newsmaker of the Year

Nature also had an end-of-2010 special article, “The Newsmaker of the Year”, in its 23/30 December issue. Unfortunately, Nature chose a U.S. government official as its Newsmaker of the Year.

We would prefer that Nature stick to what it does so very well, and stay out of U.S. politics, whether in its “opinionated editorials” [sic] or elsewhere. Perhaps the low point in Nature‘s political forays was its November 2010 editorial calling for what amounts to a new version of Prohibition. This is despite the ample evidence that moderate consumption of red wine (for example) is healthy for most adults. Readers would be well advised not to believe everything they read in Nature editorials.

Our nominee for Newsmaker of the Year in the life sciences is Dr. Svante Pääbo, for the reasons we discussed earlier.

Deals of the Year

Also as an end-of-year feature, the IN VIVO blog has been running a Deal of the Year competition. The nominees are grouped in three categories: M&A Deal of the Year, Alliance Deal of the Year, and Exit/Financing Deal of the Year.

Only one of the nominees had been featured in an article on our blog: the Celgene/Agios alliance (April 23, 2010).

The IN VIVO Blog invited readers to vote on the Deal of the Year in each of the three categories, by going to http://www.windhover.com/ezine/html/doty10.html. The voting closed at 12:00pm on 6 January 2011 (Eastern Standard Time).

The winners of the vote were:

M&A Deal of the Year: Celgene/Abraxis (50.31% of 1,799 votes)
Alliance Deal of the Year: Celgene/Agios (55.32% of 3,176 votes)
Exit/Financing Deal of the Year: Ablexis (46.54% of 1,631 votes)

Congratulations to all the winners, especially Agios and Celgene, which were featured in our blog post.

Happy New Year!

This is our own version of an end-of-year special article, and will be our last blog post of 2010. Best wishes to all of you for a happy, productive, and innovative New Year in 2011.

_______________________________________________

Posted in About Our Blog, Animal Models, Drug development, Drug discovery, Strategy and Consulting, Synthetic biology

Knockout rats made “the good old way”

Published by Allan Haberman on October 1, 2010 | Leave a response

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.

Posted in Animal Models, Drug development, Drug discovery, Neurodegenerative diseases, Psychiatric diseases

1 2 Next →

Search