Olaparib

In Part 1 of this series, we began a discussion of a new, disruptive strategy for clinical trials of oncology drugs, which had been outlined in a Perspective by Drs. Johann S. de Bono and Alan Ashworth, and published in the 30 September 2010 issue of Nature.

This strategy, which these authors call the personalized medicine hypothesis-testing strategy, is aimed at testing targeted drugs that have been developed via biology-driven drug discovery. Such a strategy begins with a strong biological hypothesis that a particular altered molecular target is critical for the malignant phenotype of a particular cancer. Based on this hypothesis, drug discovery researchers develop both targeted drugs that are specific for these altered targets, and biomarkers that can be used to determine which patients have tumors that express the target, and thus are most likely to benefit from treatment with the drug.

Following preclinical studies, clinical researchers test the drug in patients whose tumors express the target, aiming for proof of mechanism and proof of concept in early clinical trials. This involves the use of rapid dose escalation and adaptive trial design. Following these early trials, the researchers go on to conduct Phase 3 clinical trials, aiming at registration. This strategy is designed to reduce clinical attrition and the time and cost of clinical trials, and to develop superior, targeted drugs that provide greater patient benefit (in terms of progression-free survival) than the typical new oncology drugs that reach the market.

In the de Bono and Ashworth article, the authors provide several examples of successful hypothesis-testing clinical trials using this strategy. In this blog post, we discuss three of these examples, one of which is a “classic” that should be familiar to most of you, another which we have discussed in previous articles on this blog, and a third example that is based on Drs. de Bono and Ashworth’s own research.

Imatinib (Novartis’ Gleevec/Glivec)

The “classic” example of the use of a personalized medicine hypothesis-testing strategy is the development of imatinib (Novartis’ Gleevec/Glivec).  This drug was originally designed as a specific inhibitor of the ABL tyrosine kinase, which is stuck in the activated conformation in the BCR-ABL fusion protein. BCR-ABL is the “driver” mutation in Philadelphia chromosome-positive chronic myeloid leukemia (CML). Imatinib was also found to be specific for two other tyrosine kinases, c-Kit and the platelet-derived growth factor receptor (PDGFR); these findings have led to the use of imatinib to treat other cancers, especially gastrointestinal stromal tumors (GIST). We discussed the role of Dr. Brian Druker (Oregon Health Sciences University in Portland) and Nicholas B. Lydon (then at Novartis) in the development of imatinib in an earlier blog post.

The 2001 published Phase 1 clinical trial of imatinib in CML led by Drs. Druker and Lydon, and clinician Charles L Sawyers, M.D. (Memorial Sloan-Kettering Cancer Center/Howard Hughes Medical Institute) is what Drs. de Bono and Ashworth called “a landmark paper” in the use of a personalized medicine hypothesis-testing strategy to demonstrate the efficacy and safety of a targeted oncology drug. The development of imatinib for CML was made possible by basic research that showed that the BCR-ABL fusion protein (which is generated as the result of the translocation that produces the Philadelphia (Ph) chromosome, the characteristic genetic abnormality of CML) alone was sufficient to cause CML, and that the tyrosine kinase activity of the ABL moiety of the protein was required for its oncogenic activity. Researchers then discovered a compound, imatinib, that was highly specific for BCR-ABL, c-kit, and PDGFR.

The Phase I clinical trial (which took place in 1999) was a dose-escalation trial of imatinib in 83 patients with chronic-phase CML in whom treatment with interferon-alpha had failed. The primary endpoint of the trial was the safety and tolerability of the drug; efficacy was a secondary endpoint. Imatinib was found to be well-tolerated, and a maximum tolerated dose was not identified in this trial. Complete hematological responses (defined by reductions in the white-cell and platelet counts) were seen in 53 of 54 patients who received 300 mg per day or more of imatinib; these responses typically occurred in the first four weeks after initiating treatment. Cytogenetic responses were defined by the percentage of blood cells in metaphase that were positive for the Ph chromosome, ranging from major responses (zero to 35% of Ph chromosome-positive cells) to minor responses (36-65% positive) to no response (over 65% positive). Of the 54 patients treated with doses of 300 mg or more, 29 had cytogentic responses, including 17 with major responses; seven of these patients had complete cytogenetic remissions (durable zero percent Ph chromosome positive).

Blood samples were taken to determine whether BCR-ABL tyrosine kinase activity had been inhibited by in vivo treatment with imatinib. The researchers observed dose-dependent inhibition of BCR-ABL tyrosine kinase activity. This constituted proof of mechanism of the drug, while the antileukemic activity of imatinib in the trial constituted proof-of-concept.

The researchers then conducted Phase 2 clinical trials, which confirmed and extended the results seen in Phase 1. The FDA approved imatinib in May 2001, less than three years after initiation of clinical trials. This rapid approval was made possible by the FDA granting imatinib a Fast Track designation and Accelerated Approval, which allowed approval of the drug based on Phase 2 trials using surrogate markers (in this case, cytogenetic responses).

As imatinib gained approval as frontline therapy for treatment of Ph chromosome-positive CML, resistance to imatinib became an important issue. Researchers found that this resistance was usually due to mutations in BCR-ABL that interfere with imatinib binding. Two companies therefore designed inhibitors that can bind to and inhibit these resistant BCR-ABL proteins and thus successfully treat imatinib-resistant CML–dasatinib (Bristol-Myers Squibb’s Sprycel) and nilotinib (Novartis’ Tasigna). This is an example of the use of reiterative translational studies to determine mechanisms of drug resistance, and the design of second-generation drugs to combat this resistance. This type of follow-up strategy was discussed in the de Bono and Ashworth article and in our previous blog post.

Only a few years ago, many industry commentators were of the opinion that the development of imatinib to treat CML was a unique case, and development of other personalized biology-driven drug discovery-based cancer medicines would not be successful. However, the examples discussed in the de Bono and Ashworth article (and elsewhere) show that that is not true.

Roche/Plexxikon’s PLX4032

The second example of successful use of the hypothesis-testing clinical trial strategy is the development of Roche/Plexxikon’s PLX4032 for metastatic melanoma. This compound is exquisitely specific for B-Raf carrying the V600E mutation B-Raf(V600E). This is the most common somatic mutation found in human melanomas, and is a “driver mutation” that is particularly critical for the malignant phenotype of human metastatic melanomas that carry the mutation.

We have discussed PLX4032 in three articles on this blog in 2010, published on March 2, March 10, and August 27.

As in the case of imatinib, researchers achieved proof-of-mechanism and proof-of-concept for PLX4032 in a dose-escalation Phase 1 trial in patients who were preselected for carriers of the B-Raf(V600E) mutation. The Phase 1 trial took place in 2008/2009. This was followed by an extension phase in which patients were given the maximum tolerated dose of the drug. Patients showed an 81% response rate (i.e, a partial or a complete response). The estimated median progression-free survival among all patients was over 7 months, as compared to less than 2 months in large numbers of advanced melanoma patients as determined by historical analysis. Oncologists had never seen such a dramatic response in treatment of metastatic melanoma.

PLX4032 is on an accelerated path to potential registration, and parallel Phase 2 and Phase 3 clinical trials are in progress in previously treated and previously untreated patients, respectively, all who have metastatic melanoma carrying the B-Raf(V600E) mutation.

Despite the dramatic regressions and increased survival seen in the Phase 1 trials, all the patients apparently eventually suffered relapses. As stated in the article on PLX4032 in the 30 September 2010 issue of Nature, researchers are therefore doing reiterative translational studies to determine the mechanisms of resistance to PLX4032 in cases of tumor regrowth after treatment with the drug. Proposed strategies include the development of combination therapies that include PLX4032 and other targeted drugs, immunotherapeutic agents, or chemotherapy. Given the promising efficacy and safety profile of PLX4032, researchers believe that the drug has the potential to enable the development of such combination therapies.

In conjunction with the early clinical trials of PLX4032, researchers developed a real-time polymerase chain reaction (PCR) assay to assess B-Raf(V600E) mutation status. The assay has the potential to be used as a companion diagnostic in treatment with PLX4032.  As stated in the 30 September article, researchers are assessing the reliability of the PCR assay In the ongoing concurrent Phase 2 and Phase 3 clinical trials of PLX4032.

A synthetic lethal therapeutic strategy using KuDOS/AstraZeneca’s olaparib

The third example of successful use of the hypothesis-testing clinical trial strategy is taken from Drs. de Bono and Ashworth’s own work. The therapeutic strategy in this example is fundamentally different from the cases of imatinib and PLX4032, both of which are exquisitely targeted drugs that inhibit specific mutated versions of oncogenes. Instead, this example involves the use of synthetic lethality in the design of an anticancer therapeutic strategy. Based on classic studies in yeast and Drosophila, synthetic lethality is defined as a situation in which mutation in either of two genes individually has no effect, but simultaneous mutation in both genes is lethal. In cancer, if one gene in a synthetically lethal pair is defective (and especially if this defect is involved in the malignant phenotype) targeting the other gene with a drug should be selectively lethal to the tumor cells but not to normal cells. If this works, it should result in a large therapeutic window for treatment with the drug.

Women with a germline mutation in one BRCA1 or BRCA2 allele have a high risk of developing breast and ovarian cancer; BRCA1 or BRCA2 carrier status in men also carries an increased risk of developing prostate cancer. Via the process of loss of heterozygosity, cells of carriers of loss-of-function mutations in BRCA1 or BRCA2 can lose the wild-type allele, resulting in cells that lack BRCA1 or BRCA2 function. The products of the two BRCA genes are both involved in the pathway for DNA repair via homologous recombination. Loss of a functional homologous recombination pathway results in the development of genomic instability that can lead to carcinogenesis. Moreover, since BRCA-negative tumor cells cannot repair their DNA via homologous recombination, they are dependent on an alternative pathway of DNA repair, which involves the enzyme Poly(ADP) ribose polymerase (PARP). Since the average cell must repair its DNA thousands of times a day, researchers hypothesized that BRCA-negative tumor cells should be uniquely vulnerable to drugs that inhibit PARP. In contrast, normal cells are able to utilize the homologous recombination pathway, and should not be affected by PARP inhibitors.

Alan Ashworth and his colleagues developed and published this synthetic lethality strategy for therapy of BRCA-negative breast cancer in 2005. They showed that cells deficient in BRCA1 or BRCA2 were about 1,000-fold more sensitive to a class of PARP inhibitors developed by AstraZeneca (AZ) subsidiary KoDOS Pharmaceuticals (Cambridge, MA) than cells with BRCA1 and BRCA2 function. Treatment of BRCA-deficient cells with the PARP inhibitors resulted in chromosomal instability and cell cycle arrest, followed by apoptosis. The efficacy and specificity of the PARP inhibitors for BRCA-deficient cells also carried over to in vivo studies in mouse models. These cell culture and animal studies constituted the generation of a strong hypothesis that this synthetic lethal therapeutic strategy would be useful in developing antitumor treatments for patients with BRCA-negative breast cancer.

In 2006 and 2007, Drs. Ashworth, de Bono, and their colleagues (including researchers from KuDOS and AZ) conducted a Phase 1, hypothesis-testing clinical trial of KuDOS/AZ’s potent, orally-active PARP inhibitor olaparib (AZD-2281; formerly known as KU-0059436). The study enrolled a total of 60 patients with a variety of types of solid tumors, including 22 who were confirmed BRCA1 or BRCA2 mutation carriers and one patient with a strong family history of BRCA-associated cancer but who declined mutation testing. The study was published in July 2009 in the New England Journal of Medicine. The trial was a dose-escalation study–the dose was increased from 10 mg daily for two of every three weeks to 600 mg twice daily. A reversible dose-limiting toxicity was seen in one of eight patients receiving 400 mg twice daily, and in two of five patients who received 400 mg twice daily. Based on these results, the researchers established 400 mg twice daily as the maximum tolerated dose. They then enrolled a new cohort of carriers of a BRCA1 or BRCA2 mutation; these patients received a dose of 200 mg twice daily.

As a Phase 1 trial, the primary objectives were to determine safety, adverse effects, the dose-limiting toxicity and maximum tolerated dose, and the pharmacokinetic and pharmacodynamic profiles. Once these were established, the aim was to test the hypothesis that patients’ BRCA1 or BRCA2 mutation-associated cancers would show an objective antitumor response to olaparib as a single agent. In terms of safety, adverse effects were generally mild. There were two patients deaths due to infectious disease that were deemed not to be drug related. There was also no difference in adverse effect profiles between known BRCA1 and BRCA2 mutation carriers and other patients.

The researchers established three types of biomarkers. The predictive biomarker was the presence of BRCA1 or BRCA2 loss-of-function mutations, as determined by standard sequencing methods in patients with a family history of BRCA-associated cancers. The pharmacodynamic biomarker was the inhibition of PARP enzymatic activity in peripheral blood mononuclear cells and in tumor biopsies taken before and after olaparib treatment, and the formation of double-strand DNA breaks in hair follicle tissue. The intermediate endpoint biomarker consisted of radiological determination of tumor shrinkage and biochemical tests for serum tumor markers.

Using the pharmacodynamic biomarker, the researchers showed that inhibition of PARP was over 90% in peripheral mononuclear cells in patients treated with 60 mg or more of olaparib twice daily. Determination of PARP activity in tumor biopsies before and after 8 days of treatment showed that drug treatment inhibited PARP in tumor tissue. Pharmacodynamic studies in samples of plucked eyebrow hair follicles showed that induction of formation of double-strand breaks occurred within 6 hours of olaparib treatment. These studies constitute proof-of-mechanism of olaparib in humans.

In studies to determine whether olaparib treatment induced antitumor responses, the researchers found that such responses only occurred in patients with confirmed BRCA1 or BRCA2 mutation carrier status, except for one patient who declined mutational testing but had a strong family history of BRCA mutation-related cancer. 23 patents who were confirmed or (in the one case) deemed to be BRCA mutation carriers were treated. Of these 23 patients, two could not be evaluated. Two of the remaining patients had tumors not typically associated with BRCA mutations, and neither received clinical benefits from drug treatment.

Of the remaining 19 patients (who had ovarian, breast, or prostate cancer), 12 exhibited clinical benefits from olaparib treatment, with either tumor responses (determined radiologically or via serum tumor markers) or stable disease for a period of four months or more. Nine BRCA carriers had a tumor response. Eight patients with advanced ovarian cancer had a partial response (determined by radiology), and six of these had a greater than 50% tumor response based on tumor marker assays. Of the three patients with advanced BRCA2 breast cancer, one had a complete remission lasting for over 60 weeks, and another had stable disease for 7 months. The other breast cancer patient, who had refused mutational testing, had a decline in metastases and an over 50% decline in serum tumor markers. The patient with BRCA2-related castration resistant prostate cancer has an over 50% reduction in PSA levels, and resolution of bone metastases. He had been participating in the study for over 58 weeks at the time of the cutoff date, and for more than 2 years since that date.

The above efficacy data constitutes proof-of-concept, and confirms the hypothesis that BRCA-associated cancers can be addressed by a synthetic lethal therapeutic strategy based on the use of the PARP inhibitor olaparib. Olaparib also has a satisfactory adverse effect profile, and lacks the toxicity typically seen with cancer chemotherapy. Since this Phase 1 clonal trial, AZ had taken olaparib into Phase 2 clinical trials in advanced BRCA-related breast and ovarian cancer. Olaparib has continued to demonstrate efficacy and a relatively mild adverse effect profile in these trials, as shown here and here, and as also discussed in a July 2010 Medscape article.

Dr. Ashworth and his colleagues noted that not all cancers in BRCA1 or BRCA2 carriers respond to olaparib. They hypothesize that different BRCA1 or BRCA2 mutations may result in different defects in homologous recombination, which may cause variations in sensitivity to PARP inhibition. Moreover, certain secondary BRCA2 mutations may restore BRCA function, which may cause resistance to PARP inhibition. They see the need to develop assays for homologous recombination proficiency, which might be used in reiterative translational studies to determine causes of resistance to olaparib.

Synthetic lethal therapy with PARP inhibitors such as olaparib may be applicable to other types of cancers that have defects in DNA repair by homologous recombination. These may include sporadic breast and ovarian cancers that acquire loss of function of BRCA1 or BRCA2 via somatic genetic or epigenetic events, and other sporadic cancers that develop loss of function (via somatic genetic or epigenetic events) of other proteins involved in the homologous recombination DNA repair pathway.

Dr. Ashworth and his colleagues have also shown that loss of function of DNA damage signaling proteins (e.g., ATM, ATR, CHK1, CHK2), and of Fanconi anemia proteins, can induce sensitivity to PARP inhibition. Loss of function in these pathways may be relatively common in other sporadic cancers. It will be essential to develop biomarkers for loss of function of these DNA repair proteins in order to design hypothesis-testing clinical trials to investigate the potential of olaparib (or other PARP inhibitors) to treat this broader class of cancers.

As show by these three examples–and the other examples discussed in the 30 September 2010 de Bono and Ashworth Perspective (see Box 5 in that article)–researchers have been using the personalized medicine hypothesis-testing strategy to develop exciting new oncology drugs to treat disease in specific classes of patients. However, except for the case of imatinib, all of the drugs are still in clinical trials and have not yet achieved registration, which is the real test of the success of this strategy. Moreover, as we discussed in the first article in this series, the personalized medicine hypothesis-testing strategy is a work in progress. For example, biomarker identification and qualification/validation, which is a critical need for further development and utilization of this new clinical trial strategy, is an early-stage area of science and technology. Nevertheless, the personalized medicine hypothesis-testing strategy for cancer drug development provides a means to extend biology-driven drug discovery into the clinic, to decrease the time and cost of clinical trials, and to develop anticancer drugs that should be superior to both conventional chemotherapy and to early-generation targeted drugs.

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The 30 September issue of Nature included a major emphasis on translational research in cancer. Featured articles included an editorial, a Perspective, and a research report. There was also an online “Specials” archive on translational cancer research, containing many recent research reports, all with free access to nonsubscribers.

The theme of these articles was the development of novel strategies for accelerating the translation of research on cancer biology into safe and efficacious therapies.

The Perspective, entitled “Translating cancer research into targeted therapeutics”, by British researchers Johann S. de Bono, M.D., Ph.D. and Alan Ashworth, Ph.D., outlines a novel disruptive clinical trial strategy for accelerating the translation of biology-driven oncology drug discovery into the clinic, with an early determination of proof of concept. This new strategy is designed  to ameliorate the high levels of Phase 2 and Phase 3 attrition of cancer drugs, as well as to lower the cost of clinical trials and to shorten the time from preclinical studies to the approval and marketing of oncology drugs that successfully emerge from clinical trials. It also is designed to aid in the development of therapies that provide greater patient benefit (in terms of progression-free survival) than the typical new oncology drugs that reach the market.

We have discussed clinical trial strategies of this type in two of our publications–our 2009 book-length report Approaches to Reducing Phase II Attrition (available from Cambridge Healthtech Institute, and our 2009 article published in Genetic Engineering and Biotechnology News, “Overcoming Phase II Attrition Problem” (available on our website). The de Bono and Ashworth article provides a more detailed and specific presentation of this strategy with respect to oncology drug development, and provides several examples of its successful application.

In the traditional Phase 1/Phase 2/Phase 3 format of cancer drug development, clinical studies focus on treating patient populations with advanced cancers that have not been characterized in terms of their genetics and molecular biology. The trials culminate in large, pivotal randomized Phase 3 trials that typically last several years, and are aimed at regulatory approval. In most cases, new drugs that emerge from Phase 3 trials and win approval only improve survival by a few months. Patients who participate in Phase 1 and Phase 2 clinical trials usually derive little or no benefit, and a high proportion of drugs fail in Phase 2 or Phase 3. This clinical trial format determines how well a particular drug or drug combination works for the average patient. However, that treatment might not be the best for a given individual patient.

As basic researchers have advanced the study of molecular genetic pathways of cancer, drug discovery researchers have been developing targeted therapies for cancer. These drugs–such as monoclonal antibodies (MAbs) like trastuzumab (Genentech/Roche’s Herceptin) and kinase inhibitors like imatinib (Novartis’ Gleevec/Glivec) work by modulating specific biomolecules (e.g., overexpressed or mutated oncogenic proteins) that are critical for the malignant phenotype. Population-based clinical trials of unselected patients make little or no sense in developing targeted therapies. Instead, clinical researchers need to first select groups of patients whose cancers express the biomolecule to be targeted, and preferably whose cancers are driven by that particular biomolecule. This way of thinking leads to the formulation of a new clinical trial strategy, as outlined in the Perspective.

Drs. de Bono and Ashworth call this strategy a personalized medicine (or “stratified medicine”) hypothesis-testing approach. The first step in this strategy is to develop a strong biological hypothesis that a particular altered molecular target is critical for the malignant phenotype of a particular cancer. This hypothesis is usually generated as the result of laboratory and clinical studies. Researchers need to show that blocking of the function of the altered target results in a lethal or cytostatic effect in cancer cells that express the the target, but not in normal cells that do not. It is also preferred that resistance to agents that block the target is not easily gained.

In the discovery stage of this strategy, researchers need not only to identify and optimize clinical candidate drugs that modulate the target, but also biomarkers that can be used to identify patients whose tumors express the altered target and are therefore likely to benefit from treatment. These biomarkers are called “enrichment biomarkers”, and have the potential to become predictive biomarkers. (Predictive biomarkers may also be the basis for the development of companion diagnostics). It is also important to identify pharmacodynamic biomarkers (biomarkers that can be used to determine target occupancy by the drug) and intermediate endpoint biomarkers (which can assess antitumor activity of the drug–for example, radiological assessment of tumor regression). Identification and qualification/validation of biomarkers is a work in progress, and is a critical need for further development and utilization of the personalized medicine hypothesis-testing clinical trial strategy.

Once targets and drugs that modulate them have been identified, they must be validated in animal models. The issue of the inadequacy of current mouse models of cancer–mainly xenograft models in which human cancer cell lines are transplanted into immune deficient mice–to predict drug efficacy is important both in the traditional cancer drug development strategy and in the novel strategy discussed in the de Bono and Ashworth article. We have discussed development of improved animal models for cancer drug development in an earlier blog post. de Bono and Ashworth note that there is an urgent need to develop such improved animal models. Nevertheless, as discussed in the de Bono and Ashworth article, there are examples of the successful implementation of the personalized medicine hypothesis-testing strategy of cancer drug development that have used traditional animal models in the preclinical phase.

In the personalized medicine hypothesis-testing strategy, clinical trials have the same three phases as in traditional trials. However, the trials involved stratification of patients using biomarkers, such that clinical studies are done in patients whose tumors express the target of the drug. Trial design is also more flexible and adaptive, and is contingent on obtaining key clinical data. The trials focus on determining the following as early as possible:

• Proof of mechanism: determining a dose range and dosing schedule under which the drug achieves sufficient target occupancy for long enough, using biomarker-based pharmacodynamic assays.

• Proof of concept: Determining that once sufficient target occupant is achieved, the drug exhibits antitumor activity, as determined using intermediate endpoint biomarkers.

In first-in-human clinical trials, in addition to determining safety and tolerability and evaluating pharmacokinetics and pharmacodynamics as in traditional Phase 1 trials, researchers also pursue rapid dose escalation, until proof of mechanism is achieved, using the appropriate biomarkers. Researchers then move on to proof of concept hypothesis testing, at doses and dosing schedules (ideally, the maximum tolerated dose) that are sufficient to address the target for long enough to have a biological effect. Ideally, researchers should move seamlessly from determination of proof-of-mechanism to assessment of antitumor activity, via adaptive trial design and patient selection using enrichment biomarkers.

If the above early-stage strategy results in a strong determination of proof of concept, this provides the basis for moving on to Phase 3 trials in patients selected using enrichment/predictive biomarkers, with the goal of drug registration. Such Phase 3 trials should have a higher probability of success than traditional Phase 3 trials in unselected patient populations, with less than adequate demonstration of proof of concept in Phase 2.

However, in the personalized medicine hypothesis-testing strategy, there is also the need for reiterative translational studies, between the laboratory and the clinic and back to the laboratory. Such studies should be designed as early as possible in clinical development. For example, clinical trials might allow researchers to obtain tumor samples to determine mechanisms of drug resistance. Such studies might form the basis for generating further hypotheses that are relevant to reversing drug resistance, via such means as development of combination therapies or of second-generation drugs.

The personalized medicine hypothesis-testing strategy is a work in progress. However, as we shall discuss in Part 2 of this series, there are examples of its successful implementation. And this strategy provides a means to extend biology-driven drug discovery, arguably the most successful drug discovery strategy of the past decade, into early and mid-stage clinical trials, thus increasing the probability of clinical success.

Nevertheless, it must also be emphasized that our understanding of disease biology (especially cancer biology) is limited, thus limiting our ability to successfully carry out biology-driven drug discovery in all cases. However, as our understanding of disease biology grows–in an incremental manner–as the result of basic research mainly in academic laboratories, we should be able to utilize this research to develop novel, breakthrough treatments via biology-driven drug discovery and personalized medicine hypothesis-testing clinical trials.

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.

FDA logo for illustration purposes only.

On September 23, 2010, the FDA made its decision on  what to do about the antidiabetic drug rosiglitazone (GlaxoSmithKline’s [GSK’s] Avandia), based largely on the July 15, 2010 recommendations of the agency’s Endocrinologic and Metabolic Drugs Advisory Committee. That committee had voted to keep Avandia on the market with some new restrictions, because of cardiovascular safety concerns with the drug. But the committee was deeply divided with respect to the specifics of these restrictions, and whether Avandia should remain on the market at all.

The FDA decided to restrict access to Avandia by requiring GSK to submit a Risk Evaluation and Mitigation Strategy (REMS). After implementation of the REMS, Avandia will be available to patients not already taking it only if they are unable to achieve glycemic control using other drugs and, who decide not to take pioglitazone (Takeda’s Actos) for medical reasons in consultation with their physician. Patients now on Avandia will be able to continue using the drug if they appear to be benefiting from it and they acknowledge that they understand the drug’s risks. Doctors will have to attest to and document their patients’ eligibility; patients will have to review statements describing the cardiovascular safety concerns. These new restrictions should significantly limit the use of Avandia, whose use has already declined precipitously.

FDA officials Drs. Janet Woodcock, Joshua Sharfstein, and Margaret Hamburg (who is the FDA Commissioner) published the reasoning behind their decision in the New England Journal of Medicine.

On September 23, 2010, the European Medicines Agency (EMA) also took regulatory action on rosiglitazone. This agency recommended that the drug, marketed as Avandia, Avandamet and Avaglim in Europe, be taken off the market. These drugs will no longer be available in Europe within the next few months. The EMA published a question and answer document on its action.

During August and September 2010, we published a series of three articles on this blog on the safety issues with rosiglitazone and the other marketed drug of the insulin sensitizing thiazolidinedione (TZD) class pioglitazone, as well as how the biotechnology/pharmaceutical industry might develop improved, safer, and more efficacious insulin sensitizers. These articles are:

• “Avandia squeaks through an FDA Advisory Panel. But can the pharmaceutical/biotechnology industry develop better insulin sensitizers?”

• “Can the pharmaceutical/biotechnology industry develop better insulin sensitizers? A breakthrough result in the biochemistry of PPARγ.”

• “How might the pharmaceutical/biotechnology industry develop better insulin sensitizers?”

Please read these articles if you have not already done so.

Because of the key importance of insulin resistance in the pathogenesis of type 2 diabetes, and because of the major unmet needs in treatment of this disease, we believe that it is important to work to develop novel, innovative insulin sensitizers that can overcome the deficiencies of the TZDs and hopefully fill at least some of these unmet needs. These articles outline ways in which the industry might be able to accomplish this objective.

Lorcaserin

As we stated in our August 4, 2010 blog post, 2010 was supposed to be the year in which one or more new obesity drugs would be approved by the FDA and reach the market. Three new drugs developed by small California companies–Vivus Pharmaceuticals’ Qnexa, Orexigen Therapeutics’ Contrave, and Arena Therapeutics’ lorcaserin, were up for review by the FDA. This followed a long hiatus, since the FDA has approved no anti-obesity drug since 1999.

On July 15, 2010, the FDA’s Endocrinologic and Metabolic Drugs Advisory Committee voted against FDA approval of the first of these three drugs to be reviewed, Vivus’ Qnexa (phentermine/topiramate). On September 16, 2010, the same Advisory Committee voted 9 to 5 against approval of the second drug up for review, lorcaserin (expected trade name, Lorqess).

The FDA usually follows the advice of its advisory panels, but does not always do so.

Lorcaserin is a selective serotonin receptor agonist, which is specific for the 5-HT2C serotonin receptor. This contrasts with the nonselective serotonin reuptake inhibitor and serotonin-releasing agents, fenfluramine and dexfenfluramine, which are notorious for their association with heart valve abnormalities. Lorcaserin is designed to be a more selective agent that works by a similar mechanism to dexfenfluramine or fenfluramine. The anorectic effect of fenfluramine/dexfenfluramine is due to their activity on 5-HT2C, but the adverse effects of these agents appears to be due to their activity on 5-HT2B. Therefore, lorcaserin is expected to be a safer agent that fenfluramine/dexfenfluramine.

However, like fenfluramine and dexfenfluramine, the efficacy of lorcaserin appears to be minimal. Pivotal Phase III clinical trials showed an average weight loss of 5.8% among subjects taking lorcaserin, as compared to 2.5% for the placebo group.

A Phase III clinical trial published in the New England Journal of Medicine (NEJM) in July 2010 showed that the drug caused significant weight loss and improved maintenance of weight loss as compared to placebo,  in a generally healthy obese population. Lorcaserin also improved values for such biomarkers as lipid levels, insulin resistance, inflammatory markers and blood pressure. A commentary by Arne Astrup, M.D. (University of Copenhagen, Denmark) published in the same issue of the NEJM concluded that lorcaserin appeared to have efficacy that was less than or equivalent to that of the two marketed antiobesity drugs, orlistat (Roche’s Xenical) and sibutramine (Abbott’s Meridia/Reductil). However, lorcaserin appeared to be safer than either of the two marketed drugs. (The clinical trials did not compare the drugs directly.) This it appeared to Dr. Astrup that lorcaserin might have a place in the management of obesity. However, he said that where the drug would fit in obesity management remained to be seen, and that it will be necessary to be “doubly sure” about the safety of lorcaserin, given the history of the obesity drug field.

The Advisory Committee noted that lorcaserin, although its efficacy was not great, met FDA efficacy criteria for approvable antiobesity drugs. However, some panelists thought that the study population consisted mainly of healthy obese individuals, and that in populations containing more patients with comorbidities (e.g., diabetes, cardiovascular disease) there might be a lesser degree of efficacy and/or additional safety issues. There is an ongoing study of lorcaserin in obese diabetics, which is expected to be reported by the end of 2010. However, some panelists thought that the trial population (600 patients) is too small to give meaningful results. This could mean that Arena–a company with limited resources–might need to rerun its Phase III trials in a patient population that includes more people with comorbidities.

Advisory Committee members also had various safety concerns. Animal studies indicated the potential for an increased risk of cancer, especially brain and breast tumors. A few panel members were concerned about the potential of lorcaserin to cause valvular heart disease, despite Arena’s efforts to avoid that problem via the design of the drug. Most panelists, however, saw no evidence of such a risk.

The induction of cancer in animals by a drug does not necessarily mean that the drug will cause tumors in humans. However, the animal results left many panelists uneasy. Some said that if the drug were approved, there would need to be an active surveillance program to look for possible brain and breast cancer in patients taking lorcaserin. Other panelists thought that there would need to be follow-up echocardiograms to check for valvular disease if the drug was marketed.

Many panelists felt that lorcaserin was a promising drug, but that the evidence that this drug’s benefits outweigh its risk was not there yet. Thus, as with Qnexa, lorcaserin might eventually be approved, if Arena can present additional data that can overcome Advisory Committee and FDA doubts.

The third preregistration antiobesity drug, Contrave (bupropion/naltrexone) is up for review by the same Advisory Committee in December 2010.

On September 15, 2010 (the day before it recommended against approval of lorcaserin), the Endocrinologic and Metabolic Drugs Advisory Committee voted 8 to 8 on whether sibutramine  should be allowed to remain on the market. This reflects the continuing controversies with sibutramine–the drug’s modest efficacy coupled with an increased risk of cardiovascular adverse effects, as discussed in our August 4 blog post. The eight panelists who recommended that sibutramine remain on the market stipulated that the drug’s label should carry additional warnings, and restrictions on who can prescribe the drug.

Sibutramine has already been withdraw from the market in Europe as of January 2010. The Advisory Panel recommendations on sibutamine not only put the drug’s future in doubt, but constitute an additional blow to the antiobesity drug market as a whole. The loss of sibutramine from the market–in the absence of any new antiobesity drug approvals–would leave only orlistat (Roche’s Xenical), a modestly efficacious drug with adverse effects that are unacceptable to many if not most patients.

Meanwhile, Gil Van Bokkelen, Ph.D., the Chairman and CEO of the biotech company Athersys (Cleveland, OH), told Fierce Biotech that the advisory panel’s rejection of lorcaserin should not affect the prospects for Athersys’ development of an antiobesity drug that is also a selective 5-HT2C receptor agonist. Dr. Van Bokkelen stated that he believes that the advisory panel decision was the result of some compound-specific issues with lorcaserin, and the approach that Arena took in developing that drug. He believes that these issues should not apply to Athersys’ preclinical 5-HT2C receptor agonist, which has shown more selectivity for the receptor. Moreover, he says that preclinical studies suggest that the Athersys drug may be more efficacious than lorcaserin.

An interesting strategic issue in the development and commercialization of late-stage antiobesty drugs is the role of Big Pharma. Both Arena’s lorcaserin and Orexigen’s Contrave have attracted Big Pharma commercialization partners–Eisai for lorcaserin and Takeda for Contrave. The two deals are similar. Arena granted Eisai exclusive rights to commercialize lorcaserin in the United States. Eisai paid Arena $50 million upfront, and is to pay Arena up to an additional $90 million in milestone payments, depending on FDA approval and sales of the drug. Similarly, Orexigen granted Takeda North American (U.S, Mexico, and Canada) marketing rights for Contrave; Orexigen retains copromotion rights in the United States. Takeda paid Orexigen $50 million upfront, and will pay (upon FDA approval) tiered double-digit royalties (starting at 20% and increasing to 35%) on any net sales of Contrave. The deal is estimated to be worth a potential $1 billion. In both cases, the Big Pharma partner will also share the costs of further development of these drugs.

In both agreements, the Japanese Big Pharma companies pay their biotech partners a relatively small upfront fee, and in turn receive U.S. (in the case of Contrave, North American) marketing rights. Substantial payments to the biotechs depend on FDA approval and on the drugs doing well in the market. Eisai and Takeda are thus making small initial payments for the right to market products with a high risk of failing to gain approval, but a high prospect of reward in terms of sales should they gain approval. The biotech partners gain financial support for further development of the drug, the credibility of having a Big Pharma partner, and upon approval, the strength of a partner with the resources necessary to market a primary care drug in the U.S./North America.

The Contrave deal is not Takeda’s only obesity partnership. In November 2009, Takeda entered into a worldwide agreement to codevelop and commercialize antiobesity drugs with Amylin (San Diego, CA). The agreement included Amylin’s Phase II agents, pramlintide/metreleptin and davalintide, as well as other, earlier-stage compounds from both companies’ obesity programs. Amylin received an upfront payment of $75 million, and is eligible to receive development, commercialization, and sales-based milestone payments that could exceed $1 billion. Amylin may also receive tiered, double-digit royalties on any future sales. Under the agreement, Amylin will be responsible for leading development through Phase II in the U.S., and Takeda will lead later-stage U.S. development and all development outside of the U.S. The companies will share the costs of development according to an agreed-upon formula.

In February 2010, Amylin and Takeda decided to halt development of davalintide, a second-generation amylin mimetic. In a Phase II study, the weight loss efficacy and tolerability profile of davalintide was not improved over pramlintide, Amylin’s first-generation amylin mimetic that is marketed as Symlin for treatment of diabetes. Amylin is a natural pancreatic peptide hormone that slows gastric emptying and promotes satiety. (Amylin the company was named after amylin the hormone.) Symlin is indicated to help diabetics who take insulin to improve their post-meal glycemic control; it can also help these patients to lose weight.

Also in February 2010, Amylin and Takeda announced that they had selected pramlintide/metreleptin for advancement toward Phase III development. This is a combination product containing pramlintide and metreleptin. Metreleptin is the recombinant methionine human leptin originally developed by Amgen as an antiobesity treatment. However, Amgen’s metreleptin program failed in Phase II trials because of leptin resistance. In 2006, Amgen licensed its leptin franchise to Amylin.

In 2008, Amylin researchers published a report containing evidence that administration of amylin to leptin-resistant diet-induced obese rats can restore leptin responsiveness to these leptin-resistant animals. Co-administration of amylin and metreleptin resulted in decreased feeding and increased weight loss, and that the combination treatment reduced feeding and weight to a greater extent than amylin treatment alone. The combination of amylin and leptin also resulted in increased energy expenditure.

In the same publication, the researchers also reported the results of a proof-of-concept study of pramlintide/metreleptin. In this 24-week randomized, double-blind, clinical trial in overweight/obese subjects, administration of the combination of metreleptin and pramlintide resulted in 12.7% mean weight loss (an average loss of 25 pounds), significantly more than was seen with either drug administered as a single agent.

Amylin later reported (on its website) the results of a 28-week Phase IIb study completed in late 2009 followed by a extension study to 52 weeks. Patients who continued treatment with pramlintide/metreleptin for the full 52 weeks exhibited sustained weight loss, whereas those who received placebo during the extension study regained almost all of their weight. The combination therapy appeared to be generally well tolerated.

Takeda’s obesity strategy thus combines a short-term “bet” on the approval of Orexigen’s Contrave, with the longer-term development of potentially more effective agents in collaboration with Amylin.

Now the immediate focus in the obesity drug area will be on the FDA Endocrinologic and Metabolic Drugs Advisory Committee meeting on Contrave in December, as well as what the FDA will do with the panel’s recommendations on sibutramine.

Meanwhile, as we stated in earlier blog posts, many companies have adopted the strategy of developing drugs that treat both diabetes and obesity, and developing the drugs for diabetes first. As the drugs prove themselves in the clinic, with respect to safety, antidiabetic efficacy, and effects on weight loss, companies may later develop them for obesity. Liraglutide (Novo Nordisk’s Victoza) is one such drug that has been approved for treatment of type 2 diabetes in both the United States and Europe. Novo Nordisk is now also developing the drug for obesity. However, most dual diabetes/obesity drugs are in early-stage development for diabetes. Early stage obesity drug development is mainly on hold, awaiting the regulatory approval of the three late-stage drugs now nearing NDA submission.

The apparent lack of regulatory success of Qnexa and lorcaserin will therefore be expected to keep development of most early-stage drugs for obesity on hold.

A notable exception is Zafgen’s ZGN-433, a methionine aminopeptidase inhibitor now in Phase I development, which targets the vasculature of adipose tissue. Zafgen (Cambridge, MA) goes against the conventional wisdom by dedicating itself to the development of novel antiobesity medicines, in the face of all the negativity surrounding that field. However, as we implied in our blog post on Qnexa, this negativity is mainly due to the relatively poor prospects for drugs that address appetite-control pathways in the CNS that involve common neurotransmitters. Such drugs often have unacceptable adverse effects, and may also have a low degree of efficacy due to the complex and redundant nature of CNS weight control pathways. Instead, we believe that drugs that address metabolic pathways involved in both obesity and diabetes may have a better chance of success. Zafgen’s R&D strategy involves targeting adipose tissue directly, rather than CNS appetite-control pathways.