Both the 28 June 2013 issue of Science and the 27 June 2013 issue of Nature have articles or sections that feature discussions of new ways to treat or even cure various types of leukemia.

The human interest story about T-cell immunotherapy researchers in Science

The 28 June 2013 issue of Science contains an article by Science staff writer Jennifer Couzin-Frankel entitled “The Dizzying Journey to a New Cancer Arsenal”. It focuses on researchers who have been working in the engineered T cell adoptive immunotherapy project at the Perelman School of Medicine of the University of Pennsylvania. We featured a discussion of this project, which since August 6, 2012 has involved a collaboration with Novartis, in our September 12, 2012 article on this blog.

Ms. Couzin-Frankel’s article is a human interest story which especially focuses on Carl June, MD, and how he came to work on T-cell immunotherapy. This included how cancer had touched his own life, with the death of his first wife, Cynthia, in 2001. The article also focused on patients who were successfully treated with the therapy, including biotech company scientist Douglas Olson, and Emily Whitehead, who is now eight years old and achieved remission from what had been end-stage leukemia over a year ago.

As we discussed in our September 2012 article, the Penn group has been developing adoptive immunotherapy based on autologous T cells engineered with chimeric antigen receptors (CARs). Specifically, this involved a CAR with specificity for the B-cell antigen CD19, coupled with the T cell costimulatory receptor CD137 and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. (In the Science article, CD19 is referred to by its alternative name, 4-1BB.) These engineered T cells are designed for the treatment of B-cell leukemias, such as B-cell chronic lymphocytic leukemia (CLL). As discussed both in our 2012 blog article and in the 2013 Science article, Novartis has been collaborating with the Penn group in order to industrialize production of the autologous engineered T cells and their use in treatment of patients. Via the ability of Penn to patent and license its technology, the Novartis collaboration also provides a potential means to conduct clinical trials under FDA regulation, and thus to commercialize a form of adoptive cellular immunotherapy for the first time.

Nature’s special supplement on leukemia

The 27 June 2013 issue of Nature includes an entire Nature Outlook supplement on “Leukaemia”. The supplement–or at least the portion of it that consists of articles produced under Nature’s “full responsibility for all editorial content” is available free online to all.

The general theme of the special supplement is stated in the introductory article by science writer and editor Apoorva Mandavilli “While survival rates for some types of leukaemia have improved dramatically, this family of blood cancers remains a potentially fatal disease. Research in epigenetics, immunotherapy, and cell transplants offers hope. And leukaemia is proving a testing ground for the theory of cancer stem cells — leading to knowledge that could advance cancer research overall.”

The Nature Perspective on adoptive T-cell immunotherapy by Penn researchers Levine and June

Included in the supplement is a short Perspective on CAR-based adoptive T-cell immunotherapy by Drs. Bruce L. Levine and Carl H. June of the Perelman School of Medicine at the University of Pennsylvania. It is entitled “Assembly line immunotherapy”. According to this Perspective, CAR technology [unlike the earlier tumor infiltrating lymphocyte (TIL) technology] enables researchers to ” efficiently produce large populations of T cells, approximating the mass of T cells in the human immune system”.

Drs. Levine and June further assert that by “using equipment and facilities developed for blood banks and stem-cell laboratories, and by automating production”, it will be possible to make CAR-based adoptive cellular immunotherapies (ACTs) widely available. Thus leukemia treatment may be on the brink of a revolution such as the auto industry experienced in recent years in moving from manual assembly lines to robotic automation.

Despite the issue of the pharmaceutical industry and regulatory agencies such as the FDA and the European Medicines Agency being geared to developing drugs, not individually-prepared cellular therapies, Drs. Levine and June cite the case of  organ, bone-marrow, and stem-cell transplants. These modalities were seen as exotic a few decades ago, but are now utilized in treatment of tens of thousands of people. The authors thus envision that ACT may also eventually be scaled up to treat the large numbers of patients who might benefit from this type of therapy. However, this will require innovation in regulatory agency oversight, and in the means by which the pharmaceutical industry might commercialize such individualized technologies. As we discussed in our September 2012 Biopharmconsortium Blog article, Novartis and Penn are leading the way.

Moving toward cures for chronic myeloid leukemia–Dr. Charles Sawyers’ Perspective

Another Perspective in the special supplement is authored by Charles L. Sawyers, M.D. [Chair, Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer (New York, NY), and Howard Hughes Medical Institute]. The Perspective, entitled “Combined forces”, focuses on chronic myeloid leukemia (CML). The first targeted kinase inhibitor for cancer, imatinib (Novartis’ Gleevec/Glivec) was initially approved for treatment of CML.

In our October 25, 2010 article on this blog, we discussed the classic proof-of-concept clinical trial that helped launch imatinib toward FDA approval. As we discussed in that article, Dr. Sawyers was a key leader of that trial. He was a co-recipient–with Drs.  Brian J. Druker and Nicholas B. Lydon, of the 2009 Lasker~DeBakey Award for Clinical Medical Research for his work on treatment of CML.

As we discussed in our Octotber 2010 article, imatinib is highly specific for 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], as well as two other protein kinases. CML patients who are initially successfully treated with imatinib may experience resistance to that drug. As a result, two second-generation kinase inhibitors–dasatinib (Bristol-Myers Squibb’s Sprycel) and nilotinib (Novartis’ Tasigna) were developed to target imatinib-resistant mutated BCR-ABL proteins, and thus successfully treat imatinib-resistant CML. More recently–in September 2012–as mentioned in Dr. Sawyers’ Perspective, another second-generation agent, bosutinib (Pfizer’s Bosulif), has reached the market. A still newer agent, ponatinib (Ariad’s Iclusig) was approved in December 2012, under the FDA’s Accelerated Approval Program. Ponatinib is of special interest, since it  targets the T315I mutation, which confers resistance to all the other four targeted CML drugs.

In Dr. Sawyers’ Perspective, he discusses how oncologists might use the current armamentarium of targeted drugs for CML to move toward a cure for the disease. Resistance to imatinib occurs because of selection for resistant mutants of BCR-ABL . Second-generation agents inhibit BCR-ABL kinases with these mutations, thus restoring disease remission. The current armamentarium of kinase inhibitor drugs for CML covers all known resistance mutations; however, no single drug can prevent all forms of resistance.

The current paradigm for treatment of CML has been to start with imatinib, and keep treating with that agent until the patient develops resistance to that drug and disease recurs. Then the physician treats with one of the second-generation agents, which typically produces disease remission. However, this sequential treatment can select for cells with BCR-ABL molecules that contain multiple mutations, which will be resistant to all kinase inhibitors. (See a 2007 report by Dr. Sawyers and his collaborators demonstrating the hazard of sequential therapy with imatinib followed by dasatinib.)

Because the second-generation agents dasatinib and nilotinib are more potent than imatinib, they were approved for frontline therapy of CML instead of imatinib, subsequent to the publication of Dr.Sawyers’ 2007 article. They were approved for frontline therapy because of their superior clinical outcomes in head-to-head comparisons against imatinib. (Bosutinib and ponatinib are newer, and have not yet received frontline therapy approval.) However, Dr. Sawyers counsels caution, since  dasatinib and nilotinib have been studied for only 3–4 years compared with the 8–10 years of data that have amassed for imatinib. Thus replacing imatinib with one of these agents might still result in development of resistance down the road.

Dr. Sawyers postulates that Instead of focusing on which individual drug is best as a monotherapy, it is time for researchers to consider whether it might be better to use combination therapy with multiple kinase inhibitors instead of sequential therapy. Extrapolating from the experience with single- versus multi-agent therapy for tuberculosis and HIV/AIDS, a combination of two or three ABL inhibitors with non-overlapping BCR–ABL mutation resistance profiles would almost certainly prevent the emergence of drug resistance. This is particularly true in the light of ponatinib’s success against T315I.

In a recent French study cited by Dr. Sawyers, researchers found that patients with the best responses to treatment with imatinib alone (no BCR–ABL detectable for more than two years) may no longer need any kinase inhibitor drugs at all. In this study, 40% of patients had not relapsed after 18 months. This raises the possibility that these patients may be cured of their disease.

Dr. Sawyers hypothesizes that since next-generation BCR-ABL inhibitors have greater potency in clinical trials, and since two-drug combinations are superior to monotherapies in preclinical studies, upfront therapy with either a second-generation inhibitor or with a combination therapy may result in even higher percentages of patients who experience elimination of all CML cells.

Even though these more potent treatments would be even more costly than imatinib therapy, if these treatments are curative, their long-term cost will be lower than the current treatment. Therefore, they might be both medically and economically advantageous, as well as giving cancer patients what they really want–a cure.

Meanwhile, in the 18 July 2013 issue of Nature, Drs. Natalia L. Komarova (University of California Irvine, Irvine CA) and C. Richard Boland (Baylor University Medical Center, Dallas TX) published a News and Views article discussing recently published mathematical models that predict that combination therapy is more effective than sequential treatment in preventing drug resistance in cancer. These mathematical models were developed especially for treatment of CML and the solid tumors melanoma, pancreatic cancer, and colorectal cancer. But these types of models may apply to all cancers for which targeted therapies have been or are being developed.

Moving toward cures for chronic myeloid leukemia–the Novartis 27 June 2013 white paper

Bound with the Nature Outlook supplement on leukemia–immediately following the Levine & June article on adoptive immunotherapy–is a white paper by Novartis researchers (Szczudlo et al.), entitled “The Novartis research vision and approach for treating patients with chronic myeloid leukaemia”. Unfortunately, since this “sponsor feature” was not written under Nature’s “full responsibility for all editorial content”, this white paper is treated almost as an advertisement. It is not available in the online version of Nature, or anywhere else online. Perhaps Novartis will make this valuable white paper available online in the near future. As with other published reviews in scientific journals (and unlike advertisements), this white paper is signed by its authors, and has reference citations.

The subject of the white paper is developing approaches that will enable CML patients on tyrosine kinase inhibitor (TKI) therapy to safely and effectively suspend their drug therapy, while maintaining minimal residual disease (MRD) levels that are either undetectable or below the level at which there is a risk of progression to more advanced phases of disease. Such a condition is known as “treatment-free remission” (TFR).

The research that is the focus of the Novartis white paper does not involve treatment with combination therapies, but monotherapy with nilotinib (Novartis’ Tasigna). The TFR-focused clinical trials with nilotinib are made possible not only by the potency of this agent, but also the development of new diagnostic assays for level of residual disease. Traditional diagnostics for CML have been based on achieving a “complete cytogenetic response” (CCyR). A CCyR is defined as the state in which there are so few Philadelphia chromosome positive (Ph+) cells in a patient’s blood or marrow that they are undetectable by this assay.

The new diagnostic assays involves measuring levels of BCR-ABL messenger RNA (mRNA) transcripts using a real-time quantitative polymerase chain reaction (RQ-PCR). The results of these sensitive assays are reported as major molecular response [MMR–a 3-log reduction in BCR-ABL levels from the international scale (IS) baseline; molecular response ≥ 4.0 logs (MR4); and molecular response ≥ 4.5 logs (MR4.5)].

Using these assays, researchers are participating in new Novartis-sponsored clinical studies of

1. patients who had previously been treated with imatinib, without achieving MR4.5, and who were then switched to nilotinib.
2. patients treated do novo with nilotinib.

The strategy is to maintain patients on nilotinib who have achieved MR4.5 for one year at that level, and then discontinue drug treatment. These patients continue to be monitored, and must maintain ≤ MR4 in order to remain free of nilotinib treatment. Those who exceed this threshold will be put back on nilotinib. So far, in earlier studies, patients on imatinib or niolotinib who were ≤MR4 off-drug and who then exceeded this level, when put back on their drug went back to deeper levels of molecular response to therapy, and showed no drug resistance. These clinical trial protocols therefore appear to be safe.

For more information about the above clinical trials, see ClinicalTrials.gov, clinical trial number NCT01784068 and NCT01698905. Both of these trials are recruiting patients.

The Novartis white paper does discuss a different kind of combination therapy than the ones proposed by Dr. Sawyers–combination therapy with a potent TKI such as nilotinib and an agent that specifically targets leukemic stem cells (LSCs). TKI-insensitive leukemia stem cells have been implicated in the persistence of MRD, and LSCs could contribute to the re-emergence of disease following suspension of TKI treatment.

Novartis and its collaborators are now testing TKIs in combination with Novartis’ experimental agent sonidegib (LDE225). Sonidegib is an inhibitor of the hedgehog (Hh) pathway. Aberrant activation of the Hh pathway has been implicated in the activity of LSCs and of other types of cancer stem cells. A poster session that described an in vitro study of a combination of sonidegib and nilotinib in CML was presented at a scientific meeting in 2010. Sonidegib (which is also known as erismodegib) has also been undergoing preclinical studies as a potential inhibitor of prostate cancer stem cells.

Conclusions

We recommend the 28 June 2013 Science article by Jennifer Couzin-Frankel, and the special supplement on leukemia in the 27 June 2013 issue of Nature for your late summer reading. It is heartening to see that at least some researchers are moving towards cures for various types of leukemia–with potential implications for development of cures for other types of cancer.

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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 contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

On June 28, 2012 we published an article on this blog entitled “Cancer Immunotherapy: The Star Of The 2012 ASCO Annual Meeting”. Now comes the American Society of Clinical Oncology (ASCO) 2013 Annual Meeting, which took place from May 30 to June 3, 2013.

As in 2012, cancer immunotherapy was the star of the meeting.

In our June 2012 article, we focused on experimental monoclonal antibody (MAb) drugs that target the cell surface receptors programmed cell death-1 (PD-1) and programmed cell death-1 ligand (PD-L1). PD-1 is a member of the CD28/CTLA4 family of T cell regulators. Like CTLA4, the target of ipilimumab, PD-1 is a negative regulator of T-cell receptor signals. When PD-L1, which is a protein on the surface of some tumor cells, binds to PD-1 on T cells that recognize antigens on these tumor cells, this results in the blockage of the ability of the T cells to carry out an anti-tumor immune response. Anti-PD-1 MAb binds to PD-1 on T cells, thus preventing PD-L1 on tumor cells from binding to the PD-1 and initiating an inhibitory signal. Anti-tumor T cells are then free to initiate immune responses against the tumor cells. This mechanism of action is completely analogous to that of ipilimumab, which binds to CTLA4 and thus prevents negative signaling from that molecule.

Anti-PD-L1 therapeutics bind to PD-L1 on tumor cells. Ira Mellman (vice-president of research oncology at Genentech), believes that anti-PD-L1 might have fewer adverse effects than anti-PD-1. That is because anti-PD-L1 would target tumor cells while leaving T cells free to participate in immune networks that work to prevent autoimmune reactions.

Three experimental drugs in this area of immunotherapy were a main focus at ASCO in 2013. They are:

• BMS’ anti-PD-1 agent nivolumab (BMS-936558, MDX-1106), which we had discussed in our 2012 ASCO article.
• Merck’s anti-PD-1 agent lambrolizumab (MK-3475)
• Roche/Genentech’s anti-PD-L1 agent MPDL3280A

We shall focus on these three agents in this article.

Competition between BMS’ nivolumab and Merck’s lambrolizumab

As highlighted in the 2013 ASCO meeting and in reports by industry commentators such as FierceBiotech, there is a keen race between BMS and Merck to be the first to market an anti-PD-1 agent.

At the ASCO 2013 meeting, BMS researchers and their colleagues reported that a third of the patients in a Phase 1 trial of nivolumab saw tumors shrink at least 30%. They also reported that patients with solid tumors [metastatic melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC)] showed high rates of 2 year overall survival–44% for melanoma, 32% for NSCLC, and 52% for RCC (clinical trial NCT00730639).

In a first Phase 1 study of a combination therapy of nivolumab with ipilimumab in metastatic melanoma, BMS researchers and their colleagues reported that the two agents could be administered in combination safely. Clinical activity for the combination therapy appeared to exceed that of published monotherapy data for each of the two agents, with greater or equal to 80% tumor reduction at 12 weeks in 30% (11/37) of patients. In addition to the ASCO 2013 presentation, the results of this combination therapy trial were published online in the New England Journal of Medicine.

According to Fierce Biotech, BMS has 6 late-stage studies under way for nivolumab, with fast-track status in place for melanoma, lung cancer and kidney cancer.

Meanwhile, Merck announced in a June 2, 2013 press release the presentation at ASCO 2013 of interim data from a Phase 1B study evaluating its anti-PD-1 agent lambrolizumab in patients with advanced melanoma. The data was presented by Antoni Ribas, M.D., Ph.D. (Jonsson Comprehensive Cancer Center, University of California, Los Angeles). in addition to the ASCO 2013 presentation, this study was published online in the New England Journal of Medicine.

A total of 135 patients with advanced melanoma were treated. Most of the adverse events seen in the study were low grade. The confirmed response rate across all dose cohorts was 38%. The highest confirmed response rate (52%) was seen in the cohort that received the highest dose (10 mg per kilogram every 2 weeks). Ten percent of the patients in the highest-dose group achieved a complete response, with response duration ranging from 28 days to 8 months.

Response rates did not differ significantly between patients who had received prior ipilimumab treatment and those who had not. Responses were durable in the majority of patients; 81% of the patients who had a response (42 out of of 52 total) were still receiving treatment at the time of analysis in March 2013. The overall median progression-free survival among the 135 patients was over 7 months.

According to Fierce Biotech, Merck now has four clinical studies under way for lambrolizumab, including a  Phase 2 trial in melanoma and Phase 1 trials in ipilimumab-naïve patients with triple-negative breast cancer, metastatic bladder cancer and head and neck cancer. The company, which has won breakthrough drug designation from the FDA for lambrolizumab, believes that the ongoing 500-patient Phase 2 melanoma study could provide enough positive data to win FDA approval. Merck is also preparing applications for late-stage clinical trials in melanoma and non-small cell lung cancer, which are planned to launch in the third quarter of 2013.

Roche/Genentech’s anti-PD-L1 agent MPDL3280A

Genentech researchers and their collaborators presented data on a clinical study of MPDL3280A in patients with metastatic melanoma at ASCO 2013. In addition to the ASCO 2013 presentation and abstract, The Angeles Clinic and Research Institute (Los Angeles, CA) published a press release about the study. Omid Hamid, M.D. of The Angeles Clinic and Research Institute made the oral presentation at the ASCO meeting.

This study was a Phase 1, multicenter, first in human, open-label, dose escalation study (clinical trial NCT01375842), which is still ongoing. It was primarily designed to assess  safety, tolerability, and pharmacokinetics of MPDL3280A in patients with metastatic melanoma. The drug was found to be well tolerated. 35 patients who began treatment at doses of 1-20 mg/kg and were enrolled prior to Jul 1, 2012 were evaluable for efficacy. An overall response rate of 26% (9/35) was observed, with all responses ongoing or improving. Some responding patients experienced tumor shrinkage within days of initial treatment. The 24-week progression-free survival was 35%. Several other patients had delayed antitumor activity after apparent tumor progression. Of three initial patients treated with a combination of MPDL3280A and vemurafenib (Daiichi Sankyo/Genentech’s Zeboraf, a targeted kinase inhibitor), two experienced tumor shrinkage, including 1 complete response. The researchers concluded that further assessment of MPDL3280A as monotherapy and combination therapy is warranted. A Phase 1 study (NCT01656642) of a combination therapy of MPDL3280A and vemurafenib in patients with previously untreated BRAFV600-mutation positive metastatic melanoma is ongoing.

Data was also presented at ASCO 2013 on the efficacy of MPDL3280A in other solid tumors. According to Roy S. Herbst, M.D. Ph.D., (Yale Cancer Center and Smilow Cancer Hospital at Yale-New Haven) MPDL3280A showed significant anti-tumor activity and was well tolerated in patients with such cancers as NSCLC, melanoma, colorectal cancer, gastric cancer, and RCC. 29 of 140 evaluable patients (21%) exhibited tumor shrinkage, with the highest overall responses in patients with NSCLC and melanoma. Of the 29 responders, 26 patients continued responding as of their last assessment.

Researchers have also been studying PD-L1 expression levels as a potential biomarker to identify likely responders. As outlined by Dr. Herbst, responses appeared to be better among patients with higher levels of PD-L1 expression. The response rate among PD-L1-positive patients was 36% (13 of 36 patients), compared with 13% (9 of 67 patients) who were PD-L1-negative. The role that PD-L1 expression might play as a biomarker is still being explored, including attempting to determine the best way to measure the protein and other related criteria.

In addition to the Phase 1 trial of MPDL3280A/vemurafenib combination therapy in melanoma, Genentech is sponsoring a Phase 1 trial of MPDL3280A in combination with bevacizumab (Genentech/Roche’s Avastin, an angiogenesis inhibitor that targets vascular endothelial growth factor) or with bevacizumab plus chemotherapy (clinical trial NCT01633970). Genentech is also sponsoring a Phase 2 clinical trial (NCT01846416) of MPDL3280A in patients With PD-L1-positive advanced NSCLC.

Conclusions

The field of immunotherapeutic MAbs for cancer, which target negative regulators of T-cell receptor signals, continues to advance. The approval and marketing of ipilimumab provides an important proof-of-principle for this approach. Now the field is advancing to include agents that target PD-1 and its negative regulator PD-L1. Studies of BMS’ PD-1 inhibitor nivolumab have advanced as far as Phase 3, and of Merck’s lambrolizumab as far as Phase 2. Meanwhile, Roche/Genentech’s PD-L1 inhibitor MPDL3280A has reached Phase 2.

However, the in terms of clinical trial data, it is still too early to meaningfully determine the efficacy of any of the PD-1 and PD-L1 inhibitor drugs. The meaningful data will come from randomized Phase 3 trials, based on overall survival rather than tumor response rate as in currently reported trials (with the exception of the Phase 1 results of clinical trial NCT00730639 of nivolumab described earlier, which included measures of overall survival).

Nevertheless, this is an extremely exciting field, and researchers, companies, and patient communities have high expectations of success.

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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 contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

On June 11, 2013, Agios Pharmaceuticals (Cambridge, MA) filed with the U.S. Securities and Exchange Commission for an Initial Public Offering (IPO). The company plans to raise up to $86 million through this IPO. This news was reported by Fierce Biotech, the Boston Business Journal, and Xconomy, among others.

The Biopharmconsortium Blog has been following Agios since December 31, 2009, and we have posted three additional articles since. Our newest article, posted on December 28, 2012, announced the publication of an article  in the November 19, 2012 issue of Chemical & Engineering News (C&EN) by senior editor Lisa M Jarvis, in which I was quoted. More recently, Agios posted a reprint of that article on its website, which it retitled “Built to Last”. I had used that phrase in my quote in Ms. Jarvis’ article.

Agios specializes in the field of cancer metabolism. The company is working on multiple potential targets, with the goal of dominating that field, using its strong proprietary technology platform. Its financing strategy is aimed at building a company with the potential to endure as an independent firm over a long period of time–hence “built to last”. This contrasts with the recent trend toward “virtual biotech companies”–lean companies with only a very few employees that outsource most of their functions, and that are designed for early acquisition by a Big Pharma or large biotech company. Agios’ ambition to dominate the field of cancer metabolism requires a “built to last” strategy.

As Agios’ CEO David Schenkein said in the C&EN article, “You’re never going to get that with a one-target deal”. In support of that strategy, Agios has raised over a quarter of a billion dollars in funding. This has included two rounds of venture capital funding that raised a total of $111 million, and a partnership with Celgene that brought in a total of $141 million in upfront payments. According to the Fierce Biotech article, Celgene has committed to invest in Agios’ IPO.

As of yet, Agios has no drugs in clinical trials. However, the company has several drug candidates in early development. And according to the Fierce Biotech article, Agios intends to use the proceeds of the IPO to fund its first clinical trials. One of the company’s lead candidates, AG-221, which targets mutant isocitrate dehydrogenase 2 (IDH2), may reach the clinic soon, according to the Fierce Biotech article. Another Agios compound, AG-120, which targets mutant IDH1, is expected to enter the clinic in early 2014.

Recent developments in Agios’ research

The Biopharmconsortium Blog has been reporting on Agios’ research on mutant forms of IDH1 and IDH2, and their roles in human cancer, beginning with our December 31, 2009 article. Briefly, wild-type IDH1 and IDH2 catalyze the NADP+-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate. However, mutant forms of IDH1and IDH2, which are found in certain human cancers, no longer catalyze this reaction, but instead catalyzes the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2-HG). The researchers have hypothesized that 2HG is an oncometabolite, and that developing mutant-specific small molecule inhibitors of IDH1 and IDH2 might inhibit the growth or reverse the oncogenic phenotype of cancer cells that carry the mutant enzymes.

As we reported in our December 28, 2012 article, Agios researchers and their collaborators reported a series of compounds that selectively inhibit the mutant form of IDH1. These compounds were found to lower tumor 2-HG in a xenograft model. More recently, on May 3, 2013, Agios researchers and their collaborators published two research reports in the journal Science, and the company also announced the results of these studies in a April 4, 2013 press release. According to that press release, the two reports are the first publications to show the effects of inhibiting mutant IDH1 and IDH2 in patient-derived tumor samples. These results constitute preclinical support for the hypothesis that the two mutant enzymes are driving disease, and that drugs that target the mutant forms of the enzymes can reverse their oncogenic effects.

In the first of these papers (Wang et al.), the researchers reported the development of the small-molecule compound AGI-6780 (a tool compound, not a clinical candidate), which potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. AGI-6780 is an allosteric inhibitor of this mutant enzyme. Treatment with AGI-6780 induced differentiation of two IDH2-bearing tumors in vitro: a TF-1 erythroleukemia genetically engineered to express IDH2, and primary human acute myelogenous leukemia (AML) carrying the IDH2 mutation. These data provide proof-of-principle that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for AML and other IDH2-driven cancers.

In the second paper (Rohle et al.), Agios researchers and their collaborators focused on a selective mutant IDH1 (R132H-IDH1) inhibitor, AGI-5198 (also a tool compound), which is one of the mutant IDH1 inhibitors that we referred to in our December 28, 2012 article. The researchers studied the effects of AGI-5198 on human glioma cells with endogenous IDH1 mutations. AGI-5198 inhibited, in a dose-dependent manner, the ability of the mutant IDH1 to produce 2-HG. Under conditions of near-complete inhibition of 2-HG production, AGI-5198 induced demethylation of histone H3K9me3 in chromatin, and also induced expression of genes associated with differentiation to glial cells (specifically astrocytes and oligodendrocytes). Blockade with AGI-5198 also impaired the growth of IDH1-mutant—but not IDH1–wild-type—glioma cells. Oral administration of AGI-5198 to mice with established R132H-IDH1 glioma xenografts resulted in impaired growth of the tumors. Treatment of mice with AGI-5198 was well-tolerated, with no signs of toxicity during 3 weeks of daily treatment.

It is possible that Agios’ IDH1/2 inhibitors do not inhibit tumor growth by inducing differentiation, at least in the case of AGI-5198 in glioma. Rohle et al. noted that although high-dose (450 mg/kg) AGI-5198 induced demethylation of histone H3K9me3 and induced gliogenic differentiation markers, a lower dose of AGI-5198 (150 mg/kg) did not. Nevertheless, the lower dose of AGI-5198 resulted in a similar tumor growth inhibition as did the the higher dose. This suggests that in glioma cells, mutant IDH1 regulates cell proliferation and cell differentiation via distinct pathways. These pathways may have different sensitivities to levels of 2-HG, with the differentiation-related pathway requiring increased inhibition of levels of 2-HG than the proliferation-related program.

Is differentiation therapy with IDH1/2 inhibitors sufficient to provide efficacious treatment of AML and/or glioma?

A companion Perspective, authored by Jiyeon Kim and Ralph J. DeBerardinis (Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX), was published in the same issue of Science as Wang et al and Rohle et al. Kim and DeBerardinis note that the selective mutant IDH1 and IDH2  inhibitors produced cytostatic rather than cytotoxic effects. Specifically, they induced cancer cell differentiation rather than cell death.

It is possible that inducing a permanent state of differentiation may be sufficient for therapeutic efficacy. However, the survival (in a differentiated, nontumor state) of viable cells still containing potentially oncogenic mutations may eventually give rise to cancer. Therefore, it is important to determine whether the therapeutic effects of these compounds will persist over long periods of time.

In discussing AGI-6780 as a differentiation therapy in hematopoietic malignancies, Wang et al. compared their results to the action of all-trans retinoic acid (ATRA) on acute promyelocytic leukemia (APL). ATRA has be used to treat APL, and it apparently works via relieving a block in differentiation present in these leukemic cells. The use of ATRA in APL has thus been taken as a paradigm of differentiation therapy, and it is used as such a paradigm by Wang et al.

We discussed the case of ATRA treatment of APL in our April 15, 2010 article on this blog. APL patients whose leukemia is due to a PML-RARα translocation in their promyelocytes (who constitute the vast majority of APL patients) initially respond to differentiation therapy with ATRA, but eventually develop resistance to the drug. Combination therapy of ATRA and arsenic trioxide (As 2O 3) cures the majority of patients, rendering a cancer that was once uniformly fatal 90% curable. As discussed in our 2010 article, this was first modeled in transgenic mice, and then applied to human patients. APL patients whose leukemia is due to a PLZF-RARα translocation in their promyelocytes are unresponsive to both ATRA and As 2O 3. However, as discussed in our 2010 article, the corresponding mouse model does respond to a combination of ATRA and a histone deacetylase (HDAC) inhibitor such as sodium phenylbutyrate.

When this combination therapy was tested in one patient in 1998 (presumably the first patient in a clinical trial), she achieved a complete remission. Presumably, clinical trials of newer, approved HDAC inhibitors [e.g., suberoylanilide hydroxamic acid (SAHA), Merck’s Vorinostat] in combination with ATRA could be carried out.  (The SAHA/ATRA combination has been tested in a mouse model of PLZF-RARα APL.)

As in the case of Agios’ AGI-5198, ATRA may work in part via a different mechanism than induction of differentiation in APL. This is despite this case being taken as a paradigm of differentiation therapy. We referred to this briefly in our April 19, 2010 blog post. ATRA appears to produce cancer cell growth arrest at least in part via inducing degradation of the PML-RARα fusion protein. Growth arrest and differentiation appear to be uncoupled in the case of the action of ATRA on PLZF-RARα-bearing cells. [The issue of the uncoupling of RARα transcriptional activation (which induces differentiation) and RARα degradation was investigated further in a study published in April 2013.]

Is it possible–as in the case of ATRA in APL–that Agios’ therapies for targeting mutant forms of IDH1/2 will require combination with another agent to achieve long-term therapeutic efficacy? Only clinical trials can answer this question. However, perhaps it might be possible to design animal models to test this issue, and to use these models to identify agents that may be productively used in combination with the IDH1/2 inhibitors.

Conclusions

Agios IPO comes amidst a boom in biotech IPOs–especially Boston biotech IPOs. In addition to Agios, recent Boston-area IPOs include Epizyme (Cambridge, MA), TetraPhase Pharmaceuticals (Watertown, MA) and Enanta Pharmaceuticals (Watertown, MA). According to a June 14 2013 article in the Boston Business Journal, bluebird bio (Cambridge, MA) is also expected to complete its IPO during the week of June 17, 2013. We discussed bluebird bio in our October 11, 2012 Biopharmconsortium Blog article.

As with Agios, neither Epizyme, TetraPhase, Enanta, nor bluebird has any revenues from approved and marketed therapeutics. However, unlike Agios, all of these four companies have drug candidates that have reached the clinic. In addition, TetraPhase and Enanta have compounds that have completed Phase 2 clinical trials, and thus have presumably achieved proof-of-concept in humans. Thus the stock of these two companies appear to be lower risk investments than that of Agios, despite Agios’ very compelling scientific and strategic rationale. At least until its compounds achieve proof-of-concept in human studies, investing in Agios is mainly for sophisticated investors who have a high tolerance for risk. ____________________________________________________

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 contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

The Biopharmconsortium Blog has from time to time been following novel developments in anti-aging medicine, including attempts to develop activators of sirtuins. However, we have not had an article on sirtuins since December 1, 2010. At that time, we reported on the discontinuation by GlaxoSmithKline (GSK) of its lead sirtuin activator, SRT501, a proprietary formulation of the natural product resveratrol (which is found in red wine).

Sirtuins are nicotinamide adenine dinucleotide (NAD+)–dependent protein deacetylases, which have been implicated in control of lifespan in yeast, the nematode Caenorhabditis elegans, and the fruit fly Drosophila. Mammalian sirtuins have been implicated (via animal model studies) in protection against aging-related diseases such as metabolic and cardiovascular diseases, neurodegeneration, and cancer.

As we discussed in our December 1, 2010 article, GSK acquired the sirtuin-pathway specialty company Sirtris (Cambridge, MA) for $720 million in June 2008. This gave GSK ownership of Sirtris’ sirtuin modulator drugs. As stated in that article, although GSK discontinued development of SRT501, it was continuing  development of Sirtris’ non-resveratrol synthetic selective sirtuin 1 (SIRT1) activators, which in addition to their greater potency, had more favorably drug-like properties.

Recently, resveratrol and synthetic sirtuin activators such as those developed by Sirtris have come to be known as  “sirtuin-activating compounds” (STACs).

Sirtuin-activating compounds (STACs) under a cloud

As we discussed in our February 10, 2010 blog article, researchers at Amgen found evidence that the apparent in vitro activation of SIRT1 by resveratrol depended on the substrate used in the assay. The Amgen group found that the fluorescent SIRT1 peptide substrate used in the Sirtris assay is a substrate for SIRT1, but in the absence of the covalently linked fluorophore, the peptide is not a SIRT1 substrate. Resveratrol did not activate SIRT1 in vitro as determined by assays using two other non-fluorescently-labeled substrates.

Researchers at Pfizer also found that resveratrol and three of Sirtris’ second-generation STACs activated SIRT1 when a fluorophore-bearing peptide substrate was used, but were not SIRT1 activators in in vitro assays using native peptide or protein substrates.The Pfizer researchers also found that the Sirtris compounds interact directly with the fluorophore-conjugated peptide, but not with native peptide substrates.

Moreover, the Pfizer researchers were not able to replicate Sirtris’ in vivo studies of its compounds. Specifically, when the Pfizer researchers tested SRT1720 in a mouse model of obese diabetes, a 30 mg/kg dose of the compound failed to improve blood glucose levels, and the treated mice showed increased food intake and weight gain. A 100 mg/kg dose of SRT1720 was toxic, and resulted in the death of 3 out of 8 mice tested.

The Pfizer researchers also found that the Sirtris compounds interacted with an even greater number of cellular targets (including an assortment of receptors, enzymes, transporters, and ion channels) than resveratrol. For example, SRT1720 showed over 50% inhibition of 38 out of 100 targets tested, while resveratrol only inhibited 7 targets. Only one target, norepinephrine transporter, was inhibited by greater than 50% by all three Sirtris compounds and by resveratrol. Thus the Sirtris compounds have a different target selectivity profile than resveratrol, and all of these compounds exhibit promiscuous targeting.

Finally, as we reported in our December 1, 2010 blog article, NIH researcher Jay H. Chung and his colleagues found evidence that resveratrol works indirectly, via the energy sensor AMP-activated protein kinase (AMPK), to activate sirtuins. Since activation of AMPK increases fatty acid oxidation and upregulates mitochondrial biogenesis, this study suggested that the effect of resveratrol on AMPK may be more important than its more indirect activation of sirtuins in the regulation of insulin sensitivity.

All of these studies left Sirtris/GSK’s STACs under a cloud.

On March 13, 2013, GSK reported that it was shutting down Sirtris and its Cambridge MA facilities, just five years after its $720 million acquisition. GSK also said that it was offering transfers to the Philadelphia area for some of the 60 remaining Sirtris employees. Although GSK was closing Sirtris, it said that it remained confident in Sirtris’ drug candidates. The pharma company said that following Sirtris’ “highly successful” research on the biology of sirtuins, further development of Sirtris’ drug candidates “requires the resource and expertise available from our broader drug discovery organization.” GSK will be “exami[ing] [its] research against a variety of therapeutic conditions, with the aim of moving potential assets into the clinic within the next three to four years.”

New evidence that STACs activate SIRT1 in vitro under certain conditions

On 8 March 2013, the journal Science published a report by Sirtris founder David A. Sinclair, Ph.D. (Harvard Medical School, Boston MA) and his colleagues [from academia and from Sirtris, GSK, and from Biomol (Plymouth Meeting, PA)] that identified conditions under which STACs activate SIRT1 in vitro. This research report was accompanied by a Perspective in the same issue of Science authored by Hua Yuan, Ph.D. and Ronen Marmorstein, Ph.D. (Wistar Institute, Philadelphia, PA).

Dr. Sinclair and his colleagues hypothesized that the fluorophore tags on peptide substrates that were used in the original, successful SIRT1 activation assays might mimic hydrophobic amino acid residues of natural substrates at the same position as the fluorophore (i.e, +1 relative to the acetylated lysine that is engaged by SIRT1). Consistent with this hypothesis, the researchers found that non-fluorophore-tagged natural SIRT1 substrates with a large hydrophobic amino acid residue [i..e, tryotophan (Trp), tyrosine (Tyr), or phenylalanine (Phe)] at positions +1 and +6 or +1 were selectively activated by STACs. Examples of such substrates are peroxisome proliferator-activated receptor γ coactivator 1α acetylated on lysine at position 778 (PGC-1α–K778), and forkhead box protein O3a acetylated on lysine at position 290 (FOXO3a-K290). The PGC-1α–K778 peptide contains Tyr at the +1 position and Phe at the +6 position, and FOXO3a contains Trp at the +1 position. Substitution of these aromatic amino acids on either acetylated peptide with alanine (Ala) resulted in complete abolition of SIRT1 activity.

The researchers identified over 400 nuclear acetylated proteins that are potential SIRT1 targets that support STAC-mediated activation of SIRT1, on the basis of their structure. They tested five of these native sequences and found that three of them supported SIRT1 activation.

Kinetic analysis of SIRT1 activation by STACs in the presence of the above peptide substrates showed that the enhancement in the rate of SIRT1 deacetylation was mediated primarily through an improvement in peptide binding. This is consistent with an allosteric mechanism of activation. In allosteric regulation, an allosteric activator (in this case, a STAC) binds to a regulatory site (also known as an allosteric site) that is distinct from the catalytic site of an enzyme (in this case, SIRT1). Binding of the activator to the allosteric site results in the enhancement of the activity of the enzyme, for example by causing a conformational change in the protein that results in improved biding of the catalytic site to the substrate.

In order to investigate the nature of the hypothesized SIRT1 allosteric site, the researchers screened  for SIRT1 mutant proteins that could not be activated by STACs in the presence of an appropriate peptide substrate. As a result of these studies, the researchers identified a critical glutamate (Glu) residue at position 230 of SIRT1, which is immediately N-terminal to the catalytic core of SIRT1.  Glu230 of SIRT1 is conserved from flies to humans. Replacement of Glu230 with another amino acid, such as lysine or alanine, resulted in attenuation of SIRT1 activation by STACs, independent of the substrate used.  Structural studies identified a rigid N-terminal domain that contains Glu230, and is critical for activation by STACs.

The researchers then studied the effects of STACs on cultured cells (murine myoblasts), expressing either wild-type SIRT1 or mutant SIRT1 in which Glu230 is replaced with lysine (SIRT1-E222K, which is the murine equivalent of human SIRT1-E230K). Cells expressing the mutant SIRT1 did not respond to STACs, but cells expressing wild-type SIRT1 did. Specifically, cells expressing wild-type SIRT1 exhibited STAC-stimulated increases in ATP levels, mitochondrial mass, and mitochondrial DNA copy number, but cells expressing mutant SIRT1 did not. In STAC-treated cells, the researchers found no evidence of SIRT1-independent AMPK phosphorylation. This goes against studies discussed earlier in this article, that indicate that resveratrol works via activating AMPK. They also found no evidence for inhibition of phosphodiesterase isoforms in the STAC-treated cells. This goes against a study, published in Cell in 2012, that indicates that resveratrol ameliorates aging-related metabolic conditions by inhibiting cAMP phosphodiesterases, thus engaging a pathway that activates AMPK.

The researchers conclude that STACs act via a mechanism of direct “assisted allosteric activation” mediated by the Glu230-containing N-terminal activation domain of SIRT1. They further conclude that their findings support the hypothesis that allosteric activation of SIRT1 by STACs constitutes a viable therapeutic intervention strategy for many aging-related diseases. thus apparently vindicating the Sirtris/GSK development program.

However, the authors of the companion Perspective hypothesize that the reason that existing STACs only work with SIRT1 substrates that contain hydrophobic residues at position +1 to the acetylated lysine is because they were identified via screening with a substrate that contained a hydrophobic residue mimetic–i.e., a fluorophore tag. A new screen that is not biased in this way might possibly identify STACs that exhibit selectivity for SIRT1 substrates that contain other sequence signatures. It is possible that such STACs might be better therapeutics for certain aging-related diseases than the current STACs being investigated by Sirtris/GSK. There also remain many unknowns in the biology of SIRT1, and in the biochemistry of STACs –i.e., mechanisms by with certain STACs modulate the activity of biomolecules other than SIRT1 (e.g,, cAMP phosphodiesterases). Such issues might affect the success or failure of any program to develop STACs as therapeutic compounds.

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

In our February 28, 2013 article on the Biopharmconsortium Blog, we discussed the FDA’s February 7, 2013 Draft Guidance for Industry entitled “Alzheimer’s Disease: Developing Drugs for the Treatment of Early Stage Disease”.

This document had been distributed for comment purposes only, and the FDA has been seeking public comment on the draft guidance for 60 days following publication.

As we discussed, by issuing this Draft Guidance, the FDA added its voice to that of an ever-increasing segment of the scientific community that calls for a new focus on conducting clinical trials in early-stage Alzheimer’s disease (AD). This is in order to  focus industry R&D on developing treatments for patients whose disease is in a stage prior to the development of extensive irreversible brain damage. It is in this early stage of disease in which researchers believe that new drugs have the best chance of providing benefits to patients, by preventing further damage to the brain.

In our February 28, 2013 article, we also discussed several clinical trials being carried out by industry and academic researchers in early-stage AD. These trials should allow the scientific and medical community to answer the question as to whether treating patients with pre-AD or very early-stage AD with anti-amyloid MAb drugs can have a positive effect on the course of the disease, and slow or prevent cognitive decline.

Readers of our article may have noticed that the February 7, 2013 Draft Guidance was somewhat vague or confused. That is because there is currently no evidence-based consensus as to which biomarkers might be appropriate to support clinical findings in trials in early AD. Moreover, in “pre-AD” or very early-stage AD (i.e., before the onset of overt dementia) disease-related impairments are extremely challenging to assess accurately. Thus both measuring clinical outcomes and assessment via biomarkers in very early-stage AD are fraught with difficulty, making determination of drug efficacy very difficult.

In issuing the Draft Guidance, The FDA appeared to be seeking guidance from industry and from the academic community on how these issues might be resolved. As we said in our article, the early-stage AD trials now in progress might help the scientific and medical community, and the FDA, with issues of evaluation of biomarkers and clinical outcome measures in determining disease prognosis and the efficacy of drug treatments.

More recently–on March 13, 2013–the FDA proposed a further modification of its proposed guidelines for regulation of early-stage AD therapeutics. This was published online in an article in the New England Journal of Medicine (NEJM), entitled “Regulatory Innovation and Drug Development for Early-Stage Alzheimer’s Disease”, by Nicholas Kozauer, M.D. and Russell Katz, M.D. (As we stated in our earlier article, Dr.Katz is the director of the Division of Neurology Products in the FDA’s Center for Drug Evaluation and Research. Dr. Kozauer is a Clinical Team Lead in the same division of the FDA.)

The new proposal attempts to deal with some of the apparent confusion in the February 7, 2013 Draft Guidance, and to facilitate the development and approval of new drugs for early-stage AD. The NEJM article notes that traditional measures of AD drug efficacy at the FDA had included assessment both of improved cognition and improvements in function. Specifically, as stated by a New York Times article discussing the new FDA proposal, “cognition” refers to such mental processes as memory and reasoning (as assessed by various tests), and “function” refers to performing such day-to-day activities as cooking, dressing or bathing.

In the FDA’s March 13, 2013 NEJM article, the authors note that researchers and regulatory agencies “simply do not yet have drug-development tools that are validated to provide measures of function in patients with Alzheimer’s disease before the onset of overt dementia”. Thus, although one can test early-stage AD patients for improvements in cognition with the appropriate tests, testing for deficits and improvements in function is extremely difficult.

The authors of the NEJM article therefore suggest that it might be feasible that a drug for treating early-stage AD be approved via the FDA’s accelerated approval pathway, on the basis of assessment of cognitive outcome alone. The agency’s accelerated-approval pathway allows drugs that address an unmet medical need to be approved on the basis of a surrogate or an intermediate clinical endpoint–in this case, a sensitive measure of improvement in cognition. Drugs approved via “accelerated approval” must be subjected to postmarketing studies to verify the clinical benefit. This regulatory pathway might facilitate the approval of treatments that appear to be effective in early AD, when patients might be expected to derive a greatest benefit than after the development of overt dementia.

With respect to selection of patients for trials in early-stage AD, the authors of the NEJM article suggest that (based on “the consensus emerging within the AD research community”) clinical diagnosis of early cognitive impairment be combined with appropriate biomarkers. These biomarkers might include brain amyloid load [as measured by positron-emission tomography (PET)] and cerebrospinal fluid levels of β-amyloid and tau proteins. The FDA places a high priority on efforts by the researchers to qualify such biomarkers in clinical trial design in early-stage AD.

The author of the New York Times article, veteran science and medicine reporter Gina Kolata, says that the FDA’s new proposal could “help millions of people at risk of developing [AD] by speeding the development and approval of drugs that might slow or prevent it.”

She also says that the proposal could be a boon for the pharmaceutical industry and AD researchers. They have often been hampered by regulations that left them uncertain of how to get drugs tested and approved for early-stage AD. Not only might anti-AD therapies provide greater benefit to patients with early-stage AD than with later stage disease, but clinical trials in early-stage AD would have a greater potential for success–provided that researchers had appropriate means of determining efficacy in early-stage AD. The new FDA proposal may increase the likelihood of identifying such appropriate means.

As pointed out in the Times article, several leading AD researchers agree, with some important caveats. For example, AD researcher P. Murali Doraiswamy, M.D. (Duke University School of Medicine) said that the new proposed regulations would lead to more clinical trials, and more motivation now to invest in the AD field. However, many companies never manage to do postmarking studies required for drugs given accelerated approval, and such studies might not be randomized clinical trials as required in gaining approval of the drugs in the first place.

Sean Bohen, M.D., Ph.D. (Senior Vice President for Early Development at Genentech) was very positive about the proposed new FDA policy, but wondered how researchers could develop appropriate tests to identify subtle cognitive changes in early AD or pre-AD. Nevertheless, he said, “We have to start somewhere.”

Thus clinical trials in early-stage AD, and development of regulatory frameworks for approval and postmarketing studies of agents that emerge from these trials, remain a work in progress.

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