Biopharmconsortium Blog

Crizotinib

On Aug. 26, 2011, the FDA approved the kinase inhibitor crizotinib (Pfizer’s Xalkori, originally known as  PF-02341066) for treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC), in which tumor cells exhibit rearrangements in the anaplastic lymphoma kinase (ALK) gene. These rearrangements of the ALK gene constitute driver mutations that are critical for the malignant phenotype of lung adenocarcinomas that have the mutations.

Most ALK rearrangements in lung adenocarcinoma result from a deletion and inversion in chromosome 2, which produces EML4-ALK fusion genes. (EML4 refers to the echinoderm microtubule-associated protein-like 4 gene.) EML4-ALK rearrangements are found in about 4% to 5% of patients with NSCLC. This small percentage of lung cancer patients constitutes about 8,000 to 10,000 patients each year in the United States, and a worldwide patient population of around 40,000.

Crizotinib was approved together with a companion diagnostic, Abbott’s Vysis ALK Break Apart FISH Probe Kit, which is designed to help determine if a patient’s tumors have the abnormal ALK gene. The kit is designed to Identify all ALK gene rearrangements with fusion partners, including but not restricted to: EML4, TFG (TRK-fused gene), and KIF5B (kinesin family member 5B).

Crizotinib is the second targeted kinase inhibitor to be approved together with a companion diagnostic in recent weeks.  The first was vemurafenib (Plexxikon/Roche’s Zelboraf,  PLX4032), which we discussed extensively in this blog, and whose approval we covered in our August 19, 2011 article. Vemurafenib was approved together with Roche’s cobas 4800 BRAF V600 Mutation Test.

The discovery of crizotinib began with research at Sugen (San Francisco, CA), which had been acquired by Pharmacia which was subsequently acquired by Pfizer. The drug resulted from research aimed at discovery of a kinase inhibitor that targeted c-Met. The resulting drug, PF-02341066 (later known as crizotinib), is indeed a c-Met inhibitor, and was entered into Phase 1 clinical trials.  c-Met, or hepatocyte growth factor receptor, is a receptor kinase that has been implicated in cancer cell growth, migration, invasion, and metastasis.

Subsequent studies by Japanese researchers identified the inversion that results in the EML4-ALK fusion gene in a subset of human NSCLCs. They also showed that cultured mouse fibroblasts expressing the EML4-ALK fusion gene generated subcutaneous tumors in nude mice. The researchers hypothesized that the EML4-ALK fusion kinase would be a good therapeutic target, as well as a diagnostic biomarker for a companion diagnostic. Meanwhile,  researchers at Pfizer and the Massachusetts General Hospital found that PF-02341066/crizotinib was a multitargeted kinase inhibitor, which targets ALK in addition to c-Met. Pfizer researchers therefore began preclinical and clinical studies aimed at the commercialization of PF-02341066/crizotinib for treatment of patients with NSCLC carrying activating rearrangements of ALK.

Clinical trials of crizotinib in NSCLC patients with activating rearrangements of ALK

The safety and efficacy of crizotinib in NSCLC patients with activating rearrangements of ALK were established in two multi-center, single-arm studies, including a Phase 2 study (PROFILE 1005) and a Part 2 expansion cohort of a Phase 1 study (Study 1001). The studies enrolled a total of 255 patients with late-stage ALK-positive NSCLC. A sample of each patient’s tumor tissue was tested for ALK gene rearrangements before the patient could be enrolled in the study. The studies were designed to measure objective response rate, i.e., the percentage of patients who experienced complete or partial cancer shrinkage. Most patients in the studies had received prior chemotherapy.

In one study, the objective response rate was 50 percent with a median response duration of 42 weeks. In another, the objective response rate was 61 percent with a median response duration of 48 weeks.

The FDA based its approval of the Vysis ALK Break Apart FISH Probe Kit on data from one of the studies.

As part of the post-marketing requirements, Pfizer continues to evaluate critozinib in two confirmatory, randomized, open-label Phase 3 trials. In these trials, crizotinib is being compared with standard-of-care chemotherapy. One study is being carried out in previously treated patients with advanced ALK-positive NSCLC; the other trial is being carried out in previously untreated patients with advanced ALK-positive non-squamous NSCLC.

Crizotinib as a multitargeted ALK/c-Met kinase inhibitor

The epidermal growth factor receptor (EGFR) kinase inhibitors erlotinib (Genentech/Roche’s Tarceva) and gefitinib (AstraZeneca/Teva’s Iressa) are used for the treatment of patients with NSCLC with activating mutations in the intracellular kinase domain of EGFR. As with  crizotinib and vemurafenib, companion diagnostics are used to determine if a patient is likely to benefit from treatment with erlotinib or gefitinib. Activating mutations in EGFR are found in approximately 10–15% of Caucasian and 30–40% of Asian NSCLC patients.

As with most targeted antitumor drugs, acquired resistance to erlotinib or gefitinib develops in patients treated with these agents. The two most common mechanisms of this acquired resistance are:

• development of a secondary mutation that blocks binding of the inhibitors to EGFR kinase (responsible for about 50% of acquired drug resistance)
• amplification and/or activation of the c-Met kinase, or alternatively high-level expression of the natural ligand of c-Met, hepatocyte growth factor (HGF) (responsible for about 20% of acquired drug resistance).

As we discussed in Chapter 5 of our June 2011 book-length report Multitargeted Therapies: Promiscuous Drugs and Combination Therapies, Pfizer researchers and their academic collaborators found in 2010 that one could overcome HGF/c-Met-mediated resistance to erlotinib or gefitinib by combination therapy with an irreversible EGFR kinase inhibitor (such as PF-00299804) and a c-Met inhibitor (such as crizotinib/PF-02341066). The same researchers also developed a rationale for development of a companion diagnostic to identify patients with rare preexisting populations of cells with amplified c-Met genes. Such patients might be treated with the irreversible EGFR kinase inhibitor/c-Met kinase inhibitor combination. This would be expected to bypass the resistance that would develop after standard treatment with erlotinib or gefitinib alone.

Intriguingly, the 2010 Pfizer study thus suggests a second indication for crizotinib–use in combination therapy with an irreversible EGFR kinase inhibitor such as Pfizer’s PF-00299804 to overcome or preemptively circumvent HGF/c-Met-mediated resistance to the approved EGFR kinase inhibitors. However, Pfizer’s PF-00299804 is still in clinical trials, and has not yet been approved by any regulatory agency. Boehringer Ingelheim is also developing an irreversible EGFR kinase inhibitor, and Pfizer has another such agent, neratinib, in clinical trials.

Meanwhile, in addition to crizotinib, there are also other c-Met inhibitors in clinical development, including Daiichi Sankyo/ArQule’s ARQ197 and GSK/Exelixis’ XL880/GSK1363089 (now known as foretinib). ARQ197, which is in Phase 3 trials in NSCLC, is apparently the most advanced compound in development as a c-Met inhibitor.

An important potential use of irreversible EGFR kinase inhibitors is to overcome acquired resistance to first-generation EGFR kinase inhibitors in NSCLC patients due to development of a secondary blocking mutation in EGFR. The development of combination therapies of irreversible EGFR kinase inhibitors with c-Met inhibitors such as crizotinib and ARQ197 would enable their use in overcoming the second major mechanism of acquired resistance to EGFR inhibitors, via HGF/c-Met.

Conclusions

The approval of crizotinib, together with a companion diagnostic, for the treatment of ALK-driven NSCLC represents the newest example of a paradigm shift toward personalized medicine using targeted therapies in the treatment of cancer. Other examples include vemurafenib for the treatment of melanoma, and the original small-molecule targeted kinase inhibitor, imatinib (Novartis’ Gleevec/Glivec) for the treatment of chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GISTs).

In lung cancer, the use of erlotinib and gefitinib to treat EGFR-driven NSCLC, which represents about 10-15% of cases in the U.S. and Western Europe, is yet another example, even though companion diagnostics for these agents had not yet been developed at the time of their introduction to the market. ALK-driven NSCLC represents yet another 4-5% of cases.

According to researchers at the Lung Cancer Mutation Consortium, nearly 60% of patients with lung adenocarcinoma have 1 of 10 genomic abnormalities for which there is an approved or experimental drug. Paul Bunn, M.D., of the University of Colorado School of Medicine (Aurora, CO) asks, “We have 2 drugs approved now for 2 molecular abnormalities. The question is, will we go 10 for 10?”.  Diagnostic technology for testing for these mutations is also moving forward, and according to Dr. Bunn, it is cheaper to test for all ten abnormalities than it used to be to test for one abnormality.

As we discuss in our June 2011 report, and in several articles on this blog, patients treated with targeted agents usually develop acquired resistance to these drugs. Researchers, with some initial success, have been working on developing drugs to overcome this resistance. This is thus an important aspect of the development of personalized medicine for cancer.

Both EGFR-driven and ALK-driven NSCLCs are usually found in non-smokers or light smokers, while most lung cancer is associated with smoking. Physicians who treat lung cancer, as well as patients, await the development of agents that can effectively treat lung cancer in smokers and former smokers. Smoking rates have been going down in many industrialized countries, including the U.S., but that is not uniformly true in all the world. Moreover, there are still large numbers of smokers and former smokers who are at risk for smoking-induced lung cancer, and lung cancer in never-smokers (which accounts for about 10-15% of lung cancer cases) is by no means a solved problem.

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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.

Source: Narayanese. http://bit.ly/oi10H1

On July 29, 2011, Merck announced that It was shutting down the San Francisco research laboratory that it had acquired as part of its $1.1 billion acquisition of therapeutic RNAi specialist company Sirna Therapeutics. This announcement was covered in a July 29, 2011 article in Xconomy, and in a news brief in the 4 August issue of Nature and a linked Nature news blog article.

According to the Xconomy article, the shutdown will include the loss of around 50 jobs. Around ten people are being offered transfers to other Merck facilities in nearby Palo Alto CA and on the East Coast.

The Merck facility shutdown continues the exit or retrenchment from therapeutic RNAi research at other Big Pharma companies. The Biopharmconsortium Blog has covered these moves at Roche and Pfizer.

As we discussed in the Roche article, Novartis had also decided to end its 5-year partnership with therapeutic RNAi specialty company Alnylam In September 2010. However, Novartis acquired technology and exclusive development rights for RNAi therapeutics against 31 targets for in-house use as the result of its partnership with Alnylam.  Alnylam is entitled to receive milestone payments for any RNAi therapeutic products that Novartis develops based on these targets. Thus Novartis is still involved in RNAi therapeutics, despite the termination of the Alnylam partnership.

Moreover, according to the Nature news blog, Ian McConnell of Merck’s Scientific Affairs, R&D and Licensing and Partnerships said that Merck will continue to have over 100 scientists working on RNA-based therapeutics, and that it continues to invest significantly in the field. Closing the San Francisco lab represents an effort to trim the budget by eliminating the cost of maintaining a separate RNAi facility.

In our previous blog articles on Big Pharma RNAi therapeutics retrenchment, and in our October 2010 book -length report, RNAi Therapeutics: Second-Generation Candidates Build Momentum, we discussed the strategic issues that are involved in undertaking (or in retrenching from) R&D programs in RNAi therapeutics, and in investing in that area. The therapeutic RNAi (and microRNA) field represents an early-stage area of science and technology. The field may be technologically premature, as was the monoclonal antibody (MAb) drug field in the 1980s.

Big Pharma originally got into RNAi therapeutics in order to help fill weak pipelines, and with the hope of staking out a commanding position in the RNAi field once it became successful. However, with the short-term pressure at Big Pharma companies to cut expenses and programs, Big Pharmas have been losing the needed patience to continue with a technologically premature field like RNAi therapeutics.

In the June 2011 issue of Molecular Therapy, there is an editorial by Arthur Krieg, M.D. (former Chief Scientific Officer of the now-closed Pfizer Oligonucleotide Therapeutics Unit, and now Entrepreneur in Residence at Atlas Venture, Cambridge, MA), entitled “Is RNAi dead?” As discussed in the editorial, the move of Big Pharma away from RNAi, according to some observers, signals the death of the therapeutic RNAi platform. Dr. Krieg outlines an alternative view.

According to Dr. Krieg, Big Pharmas got into RNAi therapeutics with the hope of enabling the rapid development of targeted drugs without the long time lags and uncertainties of small molecule drugs and biologics. In theory, if a research team has a good target, it could rationally design a lead RNAi drug specific for the target and ready for human clinical trials within 15 months. And researchers would not have to worry about “undruggability” of targets. However, there have been several unforeseen hurdles to the development of RNAi drugs, the most formidable of which is the issue of drug delivery. Although certain high-profile publications suggested that the challenge of RNAi drug delivery could be easily overcome, this proved not to be the case in practice.

However, Dr. Krieg believes that the progress in RNAi delivery in recent years has been “nothing short of spectacular”. In 2008, the best RNAi delivery systems for a liver target might have an IC50 (i.e., the RNAi dose required for 50% inhibition of target expression) of 1–3 mg/kg, but in 2010/2011, the IC50 has been reduced to about 1% of this value, which is an improvement of two logs. Dr. Krieg also says that there have also been significant advances in reducing off-target and other undesired systemic effects of RNAi therapeutics in animal models in recent years.

Nevertheless, the advances in RNAi delivery and safety are moving too slowly for Big Pharma’s current short-term mindset. According to Dr. Krieg, if companies are not able to take an RNAi drug into clinical development this year, then the next time there is an R&D portfolio review, investments in “high-risk” technology platforms such as RNAi are likely to be cut. As we have discussed in this blog, and as is well-known to most of you, every Big Pharma company has been cutting R&D and shedding poorly productive and high-risk programs. The focus at many Big Pharmas is on fast, sure returns. High-risk or premature technologies that have not yet yielded any marketed drugs, such as RNAi (and for example, stem cells/regenerative medicine) is not likely to offer such returns.

Dr. Krieg also notes that in the case of another once-premature technology, monoclonal antibody (MAb) drugs, it took several waves of technology development to advance from repeated clinical failure to one of the most successful classes of drugs today. In our view, MAb technology is the classic case (in the life sciences, anyway) of how researchers and companies can take such a premature technology up the technology curve by developing enabling technologies. We discussed this case in our September 28, 2009 blog article, and its applicability to RNAi and stem cells in our July 13, 2009 blog article. As discussed in these articles, and as noted by Dr. Krieg, it was not Big Pharmas, but biotech companies “on the cutting edge” (together with academic labs) that advanced the therapeutic MAb field. Big Pharmas later bought into the MAb field, typically by large acquisitions. This is especially exemplified by the acquisition of MAb drug leader Genentech by Roche.

With respect to RNAi, as mentioned above, at least Merck and Novartis among the Big Pharmas are continuing with in-house RNAi therapeutics programs. And such biotechs as Alnylam, Silence Therapeutics, Quark Phamaceuticals, Dicerna, and Santaris have RNAi and/or microRNA-based drug candidates in clinical trials, often partnered with Big Pharma companies (such as Pfizer) that have cut or reduced their own RNAi drug programs. Therefore, there are companies that are working on advancing RNAi therapeutics up the technology curve. As Dr. Krieg says in his editorial, success in such programs will be expected to lead to Big Pharma reinvestment in RNAi/microRNA therapeutics, just as in the case of MAb drugs.

Two recent research reports may point the way to developing more effective, personalized therapies for two deadly women’s cancers for which their are currently few treatment options–triple-negative breast cancer and ovarian cancer. The approach followed in both reports is to use gene expression analysis to stratify each of the two diseases into subtypes. Researchers can then use gene expression and order aspects of the biology of each subtype to design subtype-specific targeted therapies, whether single drugs or drug combinations. If the drugs (whether approved or experimental) already exist, they can be tested in clinical trials, stratified by subtype. If no appropriate drugs exist, researchers can discover the drugs based on subtype-appropriate drug targets.

Triple-negative (TN) breast cancer refers to breast cancers that are negative for expression of estrogen receptor (ER), progesterone receptor (PR), and HER2. [HER2 is the target of trastuzumab (Roche/Genentech's Herceptin) and lapatinib (GlaxoSmithKline's Tykerb/Tyverb)]. Lacking all three receptors, it cannot be treated with standard receptor-targeting breast cancer therapeutics (e.g., tamoxifen, aromatase inhibitors, trastuzumab) but must be treated with cytotoxic chemotherapy. TN breast cancer is generally more aggressive than other types of breast cancer, and even treatment with aggressive chemotherapy regimens typically results in early relapse and metastasis.

TN breast cancers constitute approximately 25 percent of breast cancers. They are diagnosed most often in younger women, those who have recently given birth, women with BRCA1 mutations, and African-American and Hispanic women.

There is a Triple Negative Breast Cancer Foundation, which was founded in 2006 in honor of a mother in her mid-thirties who died of the disease.

Ovarian cancer, the ninth most common cancer in women, caused nearly 14,000 deaths in the U.S. in 2010. In its earliest stages, its symptoms are usually very subtle and mimic other, less serious diseases. As a result, it is usually detected at later stages in which treatment is more difficult and gives poorer outcomes. The 2001 five-year survival rate was 47%, up from 38% in the mid-1970s. This compared to an overall survival rate for cancer of 68% in 2001, up from 50% in the mid-1970s.

Treatment usually involves surgery and chemotherapy, and sometimes radiotherapy. Surgery (preferably by a gynecological oncologist) may be sufficient for earlier-stage tumors that are well-differentiated and confined to the ovary. In this early-stage disease (which represents about 19% of women presenting with ovarian cancer), the five-year survival rate is 92.7%. However, about 75% of women presenting with ovarian cancer already have stage III or stage IV disease, in which the cancer has spread beyond the ovaries. Then the prognosis is much poorer, and the vast majority of patients will have a recurrence.

The triple-negative breast cancer study

The TN breast cancer study was carried out by researchers at the Vanderbilt-Ingram Cancer Center (Vanderbilt University, Nashville, TN), and published in the 1 July 2011 issue of the Journal of Clinical Investigation. In this study, the researchers analyzed gene expression profiles from 21 publicly available breast cancer data sets, and identified  587 cases of TN breast cancer (by non-expression of mRNAs that encode ER, PR, and HER2). Using cluster analysis, they identified six TN breast cancer subtypes:

• two basal-like subtypes (BL1 and BL2),
• an immunomodulatory (IM) subtype (i.e., expressing genes involved in immune cell processes)
• a mesenchymal (M) subtype
• a mesenchymal stem–like (MSL) subtype
• a luminal androgen receptor (LAR) subtype.

Using gene expression analysis, the researchers identified TN breast cancer model cell lines that were representative of each of these subtypes. On the basis of their analysis, the researchers predicted “driver” signaling pathways, and targeted them pharmacologically as a proof-of-principle that analysis of gene expression signatures of cancer subtypes can inform selection of therapies.

BL1 and BL2 subtypes had higher expression of genes involved in the cell cycle and response to DNA damage, and model cell lines preferentially responded to cisplatin. M and MSL subtypes were enriched for expression of genes involved in the epithelial-mesenchymal transition (EMT), and growth factor-related pathways in model cell lines responded to the PI3K/mTOR inhibitor BEZ235 (Novartis, now in Phase 1 and 2 for solid tumors) and to the ABL/SRC inhibitor dasatinib [Bristol-Myers Squibb's Sprycel, currently approved for treatment of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL), and under investigation for treatment of solid tumors). The LAR subtype was characterized by androgen receptor (AR) signaling, and included patients with decreased progression-free survival. LAR model cell lines were uniquely sensitive to the AR antagonist bicalutamide (AstraZeneca's Casodex/Cosudex, currently approved for the treatment of prostate cancer and hirsutism, and under investigation for treatment of androgen receptor-positive, ER negative, PR negative breast cancer).

The researchers plan to use the TN breast cancer subtype-specific model cell lines for further molecular characterization, to identify new components of the “driver” signaling pathways for each subtype. These pathways can be targeted in further drug discovery efforts. The subtype-specific cell lines can also be used in preclinical studies with targeted agents, and in identification of subtype-specific biomarkers that can potentially be used in stratifying TN breast cancer patients so that they might be treated with the best agents for their disease.

The ovarian cancer study

The ovarian cancer study was carried out by the Cancer Genome Atlas Research Network [a consortium of academic researchers jointly funded and managed by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI)], and published in the 30 June 2011 issue of Nature. In this study, the researchers analyzed mRNA expression, microRNA expression, promoter methylation and DNA copy number in 489 high-grade serous ovarian adenocarcinomas, as well as the DNA sequences of exons from coding genes in 316 of these tumors. Serous adenocarcinoma is the most prevalent form of ovarian cancer, accounting for about 85 percent of all ovarian cancer deaths.

The researchers found that nearly all of the high-grade serous ovarian cancers (HGS-OvCa) studied had mutations in the TP53 gene, which encodes the p53 tumor suppressor protein. On the basis of their gene expression (mRNA) signatures, the researchers divided the population of HGS-OvCa into four subtypes:

• an immunoreactive subtype (i.e., expressing genes involved in immune cell processes)
• a differentiated subtype (high expression of markers of differentiated female reproductive tract epithelia)
• a proliferative subtype (high expression of markers of cell proliferation)
• a mesenchymal subtype (high expression of HOX genes and of markers of mesenchymal-derived cells)

The researchers also determined subtypes on the basis of microRNA expression and promoter methylation. microRNA subtype 1 overlapped the mRNA proliferative subtype and miRNA subtype 2 overlapped the mRNA mesenchymal subtype. Patients with miRNA subtype 1 tumors survived significantly longer that those with tumors of other microRNA subtypes.

Although the researchers found no significant difference in survival between the four transcriptional subtypes, they did identify a 193-gene expression signature that was predictive of overall survival. 108 genes were correlated with poor survival and 85 were correlated with good survival.

The researchers identified cancer-associated pathways in the HGS-OvCA population; this is equivalent to the prediction of “driver” signaling pathways in the TN breast cancer study. They found that 20% of the HGS-OvCA samples had germline or somatic mutations in BRCA1 or BRCA2, and that 11% lost BRCA1 expression through DNA hypermethylation. As we discussed in an earlier article on this blog, BRCA1- or BRCA2-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). These tumors are thus sensitive to a class of drugs known as PARP inhibitors, such as KuDOS/AstraZenaca’s olaparib. There are now six PARP inhibitors, including olaparib, in clinical development.

The researchers found genetic alterations in several other genes involved in homologous recombination. Altogether, defects in homologous recombination may be present in approximately half of HGS-OvCa cases, and these tumors may be sensitive to PARP inhibitors. This provides a rationale for clinical trials of PARP inhibitors in women with ovarian cancers with defects in homologous recombination-related genes.

Olaparib and other PARP inhibitors are in clinical trials in women with advanced with BRCA-1 or -2 mutations and with other defects in homologous recombination. As discussed in the 2011 ASCO meeting, early Phase 2 results indicate that olaparib gives dramatic improvements in progression-free survival in these women. (See this article and this video.) In these studies, in addition to tumors with genetic defects in homologous recombination, olaparib or another PARP inhibitor, Abbott’s ABT-888, appears to give improved progression-free survival in women who have previously been treated with chemotherapy drugs that result in DNA damage. This suggests that oncologists may be able to use a “one-two punch”, consisting of a DNA-damaging drug [such as the alkylating agent temozolomide [Merck's Temodar]) followed by a PARP inhibitor, to treat advanced ovarian cancer.

In addition to BRCA-1 and BRCA-2 mutations and other genetic alterations that result in defects in homologous recombination, the HGS-OvCa population exhibited genetic changes that would result in deregulation of several other cancer related pathways. These pathways included the RB1 (67% of cases), RAS/PI3K (45% of cases), and NOTCH (22% of cases) pathways, as well as the FOXM1 transcription factor network (87% of cases). All of these pathways represent opportunities for target identification and drug discovery. FOXM1 (Forkhead box protein M1) was named the Molecule of the Year for 2010 by the International Society for Molecular and Cell Biology and Biotechnology Protocols and Research (ISMCBBPR) because of “its growing potential as a target for cancer therapies.” FOXM1 overexpression results in destabilization of the cell cycle, which can lead to a malignant phenotype.

The researchers also identified 22 genes that were frequently amplified or overexpressed in HGS-OvCA tumors (other than genes that are involved in homologous recombination). Inhibitors (including approved and experimental compounds) already exist for the products of these genes, and researchers might assess these compounds in HGS-OvCa cases in which target genes are amplified.

Can Verastem develop new therapeutics for triple negative breast cancer?

The private biotechnology company Verastem (Cambridge, MA) focuses on discovery and development of drugs to target cancer stem cells. The company was founded in 2010, and is based on a strategy for screening for compounds that specifically target cancer stem cells. This strategy, published in the journal Cell in 2009, was developed by Drs. Robert Weinberg (MIT Whtehead Institute), Eric Lander (Broad Institute of MIT and Harvard University), and Piyush Gupta (MIT and Broad Institute) and their colleagues. Drs. Weinberg, Lander, and Gupta are on the Scientific Advisory Board of Verastem.

On July 14, 2011, Verstem announced that it had raised $32 million in a Series B financing. Verastem had previously raised $16 million from a group led by former Christoph Westphal’s Longwood Founders Fund. Dr. Westphal (formerly of Sirtris) is now Chairman of Verastem.

Cancer stem cells are best known in acute myeloid leukemia (AML), but their existence in other cancers (especially solid tumors) is controversial. The cancer stem cell hypothesis asserts that a small subpopulations of cells in a leukemia or solid tumor have characteristics that resemble normal adult stem cells, such as self renewal, the ability to give rise to all the cell types found in the leukemia or cancer, and stem cell markers. The hypothesis further asserts that most cancer treatments fail to knock out cancer stem cells, which can repopulate a tumor cell population, resulting in treatment relapses. Cancer stem cell researchers therefore propose developing cancer stem-cell specific therapeutics that can be used to eliminate these cells, which can block these relapses.

Whether cancer stem cells are involved in the pathobiology of solid tumors or not, the biology of the putative cancer stem cell phenotype can be important in certain subtypes of cancer. Cancer stem cells are characterized by the epithelial-mesenchymal transition (EMT), and in the Cell paper the researchers screened for compounds that specifically targeted breast cancer cells that had been experimentally induced into an EMT, and which as a result exhibited an increased resistance to standard chemotherapy drugs.   They identified the compound salinomycin as a drug that specifically targeted these cells, as well as putative cancer stem cells from patients.

As discussed earlier in this article, TN breast cancer includes two subtypes that have gene expression signatures related to the EMT: the mesenchymal (M) subtype and the mesenchymal stem–like (MSL) subtype. One or both of these subtypes might be sensitive to compounds that specifically target putative breast cancer stem cells. This may be true whether the cancer stem cell hypothesis applies to TN breast cancer or not. Verastem recognizes this, and is thus focusing on TN breast cancer as its first therapeutic target. The Vanderbilt TN breast cancer study suggests that trials of any “cancer stem cell-specific” therapeutics for TN breast cancer should be guided by subtype-specific biomarkers.

Hope for treatment of TN breast cancer and advanced ovarian cancer

Researchers and oncologists have made great strides in increasing the percentage of breast cancers that are treatable or even curable in recent years. For example, prior to the FDA approval of trastuzumab in 1998, HER2 positive breast cancer carried a grim prognosis. But the advent of trastuzumab (and later, lapatinib) has had a major impact on treatment of this once uniformly deadly type of breast cancer.

We hope that the new, personalized medicine-based approach to TN breast cancer and advanced serous ovarian adenocarcinoma will also result in successful new therapeutic strategies for these deadly women’s cancers.

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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.

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.

Huntington's disease. Dr. Steven Finkbeiner. http://bit.ly/q48xdX

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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.

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.

On June 1, 2011, Cambridge Healthtech Institute’s (CHI’s) Insight Pharma Reports announced the publication of our new book-length report, Multitargeted Therapies: Promiscuous Drugs and Combination Therapies.

In the past 20 years or so, pharmaceutical and biotechnology industry R&D has been increasingly aimed at developing drugs to treat complex diseases such as cancer, cardiovascular disease, type 2 diabetes, and Alzheimer’s disease. However, the one drug-one target-one disease paradigm that has become dominant in the post-genomic era has proven to be inadequate to address complex diseases, which have multiple “causes”, and each of which may be more than one disease. This has been a major cause of clinical failure and the low productivity of the pharmaceutical industry.

Moreover, researchers have found that most of the successful, FDA-approved small-molecule drugs that were developed prior to the year 2000 are promiscuous, i.e., they are single drugs that address multiple targets. In addition, the great majority of kinase inhibitors, one of the most successful drug classes of the early 21st century, are also promiscuous.

The study of small-molecule drug promiscuity has spawned the emerging field of network pharmacology, which can be applied both to study drug promiscuity and to rationally design small-molecule multitargeted drugs. (Researchers can discover or design multitargeted kinase inhibitors without the use of network pharmacology, however.)

Meanwhile, the development of targeted drugs such as kinase inhibitors and monoclonal antibodies has resulted in the need to develop multitargeted combination therapies. This has been especially true in cancer, where disease causation may involve multiple signaling pathways. In particular, the development of resistance to targeted antitumor drugs has spawned the need to develop second-generation treatments, many of which are multitargeted combination therapies.

Our report covers both discovery and design of small-molecule promiscuous/multitargeted drugs, and of multitargeted combination therapies.

The design of multitargeted combination therapies is one of the hottest areas of cancer R&D today, especially with respect to developing means to overcome resistance to targeted therapies. This area was the focus of many key presentations at the 2011 American Society of Clinical Oncology (ASCO) Annual Meeting, which was held in Chicago on June 3-7. For example, treatment with vemurafenib (PLX4032) of metastatic melanoma patients whose tumors carry the B-Raf(V600E) mutation has produced spectacular overall response rates and increased survival. However, in nearly all cases, the tumors relapse. The latest results with vemurafenib were discussed at ASCO 2011, as well as strategies to overcome resistance to therapy. Our new report also discusses strategies for overcoming vemurafenib resistance, all of which involve design of multitargeted combination therapies.

Another topic discussed at ASCO 2011 was antitumor strategies based on synthetic lethality. We discussed this strategy in an earlier article on this blog, especially with respect to poly(ADP) ribose polymerase (PARP) inhibitors such as KuDOS/AstraZenaca’s olaparib. At a session at the ASCO meeting entitled “PARP Inhibitors, DNA Repair, and Beyond: Theory Meets Reality in the Clinic”, speakers reviewed current progress in developing PARP inhibitors, of which six are now in clinical development.

This session also included a presentation by Michael B. Kastan, MD, PhD (St. Jude Children’s Research Hospital, Memphis TN) on other ways of using the synthetic lethally strategy, for example by targeting kinases involved in DNA repair pathways such as ATM (Ataxia-Telangiectasia Mutated) or Chk1 checkpoint kinase, or even utilizing features of the tumor microenviroment such as hypoxia. Such strategies might be used to design multitargeted combination therapies that specifically target cancer cells with defects in DNA repair and/or in hypoxic solid tumors, and/or to sensitize cancer cells to radiation.

Our new report includes a chapter on using the synthetic lethality strategy to design combination therapies of a cytotoxic drug with a chemosensitizing agent, and to develop therapies for p53-negative cancers. (The key tumor suppressor p53 is deleted, mutated, or inactivated in the majority of human cancers).

Although design of multitargeted combination therapies, as well as discovery and design of kinase inhibitors, are of key importance for current oncology R&D and are also being applied to other diseases, design of single small-molecule multitargeted drugs via network pharmacology is an early-stage, and perhaps a premature, technology. Nevertheless, given the current pharmaceutical company R&D business model that emphasizes outsourcing early-stage R&D, academic research groups and biotechnology companies that are active in this area may be able to forge partnerships with pharmaceutical companies.

For more information on Multitargeted Therapies: Promiscuous Drugs and Combination Therapies, or to order it, see the Insight Pharma Reports website.

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.

Galega officinalis (Goat's Rue) From JoJan http://bit.ly/l5Ybco

Metformin (Bristol-Myers Squibb’s Glucophage, generics), an oral biguanide antidiabetic drug, is the most widely prescribed agent for treatment of type 2 diabetes. The drug mainly works by lowering glucose production by the liver, and thus lowering fasting blood glucose.

Although metformin–approved in the United States in 1994, and in Europe prior to that–has been used for many years, its mechanism of action is not well understood. In 2005, signal-transduction pioneer Lewis Cantley (Beth Israel Deaconess Cancer Center/Harvard Medical School, Boston MA), and his colleagues–including Reuben J. Shaw (now at the Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA)–published a report showing that metformin targets the adenosine monophosphate (AMP)-activated kinase (AMPK) pathway in the liver. We discussed this report and its implications in our 2007 Cambridge Healthtech Institute Insight Pharma Report, Diabetes and Its Complications.

AMPK is found in all eukaryotic organisms, and serves as a sensor of intracellular energy status. In mammals, it also is involved in maintaining whole-body energy balance, and helps regulate food intake and body weight. We  have discussed the potential role of AMPK in regulation of lifespan, and as a target in anti-aging medicine and in metabolic disease, in earlier articles on this blog. (See here and here.)

AMPK is activated by increases in the ratio of AMP to ATP, caused by energy stress. Under conditions of energy stress, AMP levels go up, and AMP binds to a specific site on the AMPK γ subunit. This induces a conformational change that exposes the activation loop of the α subunit. This allows an upstream serine/threonine kinase to phosphorylate this activation loop. In several mammalian cell types, including liver and skeletal muscle, that kinase is LKB1. Drs. Cantley and Shaw in 2005 showed that metformin targets the LKB1-AMPK pathway in the liver, and that metformin requires LKB1 to lower glucose production by the liver. However, neither LKB1 nor AMPK is the direct target of metformin, and as of 2005, that direct target was unknown.

A new genetic study that suggests that ATM kinase may affect the ability of patients to respond to metformin

Now–as of February 2011–comes a Nature Genetics paper that indicates that the serine/threonine kinase ATM (ataxia telangiectasia mutated) acts upstream of AMPK to mediate the therapeutic effects of metformin. ATM is a DNA repair protein that is recruited and activated by double-strand breaks in DNA. It initiates activation of the DNA damage checkpoint, leading to cell cycle arrest, followed by DNA repair or apoptosis. Thus the role of ATM in the AMPK pathway and in the therapeutic effects of metformin is surprising indeed.

In the study reported in the Nature Genetics paper, researchers of The GoDARTS and UKPDS Diabetes Pharmacogenetics Study Group and The Wellcome Trust Case Control Consortium 2 performed a genome-wide association study (GWAS) for glycemic response to metformin in type 2 diabetes patients in the U.K. In a population of nearly 4,000 patients, they identified a single-nucleotide polymorphism (SNP) designated rs11212617, which was associated with treatment success. This SNP occurs in a genetic locus that also contains the gene that encodes ATM. In a rat hepatoma cell line, inhibition of ATM by the specific inhibitor KU-55933 (KuDOS Pharmaceuticals, Cambridge, U.K., which was acquired by AstraZeneca in 2005) attenuated metformin-mediated phosphorylation and activation of AMPK.

The analysis by Morris Birnbaum and Reuben Shaw in the 17 February 2011 issue of Nature

The 17 February 2011 issue of Nature contained a Forum entitled “Genomics: Drugs, diabetes and cancer.” This consisted of two analyses of the implications of the Nature Genetics paper for metformin’s mechanism of action, and for understanding diabetes and the connections of the metformin-activated ATM/AMPK pathway with cancer. The first analysis was by Morris J. Birnbaum, M.D., Ph.D. (University of Pennsylvania Medical School, Philadelphia, PA), who does research on the role of AMPK and insulin in energy metabolism and in diabetes. The second analysis is by Dr. Reuben Shaw, mentioned earlier. Dr. Shaw’s research centers around LKB1 [also known as serine/threonine kinase 11 (STK11)]. LKB1, a serine/threonine kinase, is not only a regulator of hepatic glucose production via AMPK, but is also a tumor suppressor. Germline mutations in LKB1 are associated with the familial cancer Peutz-Jegher syndrome, and somatic mutations in LKB1 are present in various other cancers. In particular, the Lkb1 gene is one of the most frequently muted genes in human lung adenocarcinomas.

Dr. Birnbaum’s analysis

Dr. Birnbaum notes that the finding of a role for ATM in metformin responsiveness may be an important clue to the mechanism of action of this drug. However, it may also be a false lead, with ATM having only an indirect effect on metformin’s action. He cites recent evidence that metformin acts independently from LKB1 and AMPK and of transcriptional regulation in general. In these studies, genetic ablation of LKB1 and AMPK was used to show that these mediators are dispensable for metformin’s glucose-lowering activity. Instead, metformin appears to work by inhibiting mitochondrial production of ATP in the liver, thus reducing the level of liver glucose production via gluconeogenesis (which uses ATP). This is in apparent contradiction to the 2005 results of Dr Shaw and his colleagues. Nevertheless, metformin’s inhibition of mitochondrial ATP production increases the ratio of AMP to ATP, and thus activates AMPK. There are also other pathways by which inhibition of mitochondrial ATP production may inhibit gluconeogenesis. Thus the mechanisms by which metformin causes a decrease in glucose production by the liver appear to be very complex, and are not well understood.

Dr. Birnbaum therefore speculates that ATM may affect blood glucose levels via pathways that are parallel to, but not the same as, those modulated by metformin. However, the effects of these other pathways may be synergistic with those modulated by metformin when patients are treated with the drug. Dr. Birnbaum notes that 40 years ago, it was found that patients with ataxia telangiectasia often display a type 2-diabetes-like condition, including insulin resistance. Ataxia telangiectasia is a familial disease caused by germline mutations in ATM. This suggests that  ATM may act to counteract hyperglycemia and insulin resistance.

Dr. Birnbaum concluded that biochemical and cell biology studies should be conducted to determine the nature of the interaction of ATM and the antidiabetic effects of metformin. Key to these endeavors is to determine whether there are any biomolecules other than AMPK that both are influenced by ATM and control metabolism.

Dr. Shaw’s analysis

Dr. Shaw first discusses several animal studies that help elucidate the role in glucose regulation of the biomolecules involved in the putative ATM-LKB1-AMPK pathway. He notes notes that deletion of the Lkb1 gene in mouse liver results in loss of AMPK activity in that organ, and to the development of hyperglycemia and hepatic steatosis–two conditions that are seen in type 2 diabetes. Dr. Shaw also cites the 40-year-old finding about the connection between  ataxia telangiectasia and insulin resistance and diabetes. But as he also mentions the more recent (2006) finding that mice with defective ATM activity show increased insulin resistance and abnormal glucose regulation.

Dr. Shaw then speculates as to how ATM might work to modulate patients’ antidiabetic responses to metformin. He notes that ATM is known to phosphorylate LKB1, which is the key activator of AMPK in the liver. Alternatively, ATM might also regulate AMPK independently of LKB1, and might affect responsiveness of patients to metformin by regulating other relevant targets, independently of AMPK. In this context, ATM is known to phosphorylate other, LKB1 and AMPK-independent components of the insulin signaling pathway.

In the light of these considerations, Dr. Shaw says that it is important to determine whether the rs11212617 genetic variant results in modulation of ATM activity toward AMPK activation or toward other targets relevant to glucose regulation, or indeed whether this SNP affects ATM activity at all.

Dr. Shaw then focuses on the potential relevance of metformin to cancer therapy. Researchers have found, in retrospective studies, that diabetes patients who take metformin have a lower risk of developing cancer than those treated with other antidiabetic medications. Animal studies confirm the anticancer effects of metformin, but–as discussed in a 2010 review by Dr. Michael Pollak (McGill University, Montreal, Quebec, Canada)–they indicate that the anticancer effects of this drug are mechanistically complex. Dr. Shaw asks whether metformin is a general activator of ATM (and/or its targets) in the DNA damage-response pathway, or whether its specific effects on LKB1 and/or AMPK might be responsible for the apparent beneficial effects of metformin on cancer risk.

Dr. Shaw concludes with the statement that future studies of the relationship between metformin action, ATM, LKB1, and AMPK should shed light on the relationship between metformin’s antidiabetic effects and its apparent anticancer effects.

Our conclusions

The finding, based on a genome-wide association study, which suggests that ATM, a kinase best known for its involvement in DNA repair pathways, may also be involved in diabetics’ response to metformin is surprising and intriguing. It may eventually be important in unraveling metformin’s mechanism of action in inhibition of liver gluconeogenesis, and in other antidiabetic activities. This finding indicates a connection between pathways by which metformin exerts its antidiabetic activities, and pathways that are involved in cancer.

Nevertheless, the elucidation of metformin’s mechanism(s) of action in diabetes remains a work in progress. This situation is an example of how science works in the real world (as opposed to textbooks or much of science journalism)–generating more questions than answers.

A drug like metformin, with its complex and still poorly understood mechanism of action, could not have been discovered by modern, post-genomics drug discovery strategies. Metformin was discovered via research on natural products derived from the plant Galega officinalis (known as the French lilac, goat’s rue, and by various other names), which had been known by herbalists for centuries. It is fortunate that researchers were able to study the effects of extracts of this plant, and ultimately to develop metformin, well in advance of the modern era of drug discovery. Diabetics and their physicians now have access to metformin as an inexpensive generic drug.

The continued study of the antidiabetic mechanism(s) of action of metformin may yield additional insights into control of gluconeogenesis and other metabolic pathways. Some of the findings of these studies might be relevant to drug discovery and development, for example the development and use of AMPK activators in metabolic disease and in anti-aging medicine.

Continued study of the mechanism(s) of action of metformin may also be relevant to developing new therapies for cancer. As suggested by Dr. Pollak, although metformin is off-patent and is thus not an attractive agent for development as an oncology drug by pharmaceutical or biotechnology companies, other biguanides or related compounds might be better anticancer compounds, and would be patentable. In addition to identifying such compounds, it will be important to determine and define which groups of cancer patients could best benefit from them (perhaps via biomarkers). It will then be important to conduct personalized medicine hypothesis-testing clinical trials (as discussed in an earlier blog post) designed to obtain proof-of-concept that such compounds can indeed benefit specific groups of patients.

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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.

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.

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