PPAR complex coactivator interaction

This is part 3 of a three-part series on insulin sensitizers for treatment of type 2 diabetes.

In part 1 of the series (posted August 23, 2010), we focused on safety issues with the two marketed thiazolidinedione (TZD) peroxisome proliferator-activated receptor gamma (PPARγ) agonists–rosiglitazone and pioglitazone (Takeda’s Actos). Both of these insulin sensitizing, antidiabetic agents induce weight gain, and carry an increased risk of edema and heart failure. In addition, rosiglitazone carries an increased risk of myocardial infarction. Because of the latter risk, some critics would like to see it removed from the market. However, on July 15, 2010, by a close vote the FDA’s Endocrinologic and Metabolic Drugs Advisory Committee voted to leave rosiglitazone on the market, with some new restrictions.

In part 2 of the series (posted on August 29, 2010, we discussed a breakthrough discovery by Bruce Spiegelman (Dana-Farber Cancer Institute and Harvard Medical School, Boston MA) and his colleagues, published in the 22 July issue of Nature. It was the Spiegelman group that originally identified PPARγ as the master regulator of adipocyte biology and differentiation, which eventual led to the development of the TZD drugs.

In the new research, the Spiegelman group found evidence that the insulin sensitizing and antidiabetic effects of PPARγ agonists may not be due to the agonistic effects of these compounds on PPARγ, but to their ability to inhibit phosphorylation (at Ser 273) of PPARγ by the enzyme cyclin-dependent kinase 5 (CDK5). A weak PPARγ agonist, the benzoyl 2-methyl indole (non-TZD) MRL24, inhibits CDK5 phosphorylation of PPARγ as well as rosiglitazone, and also has very good antidiabetic activity. MRL24 had been discovered by Merck in 2005.

CDK5 phosphorylation of PPARγ does not change the ability of PPARγ to upregulate transcription of genes involved in adipocyte differentiation. However, it inhibits the ability of PPARγ to upregulate transcription of a set of genes, including the gene for the adipokine adiponectin, that induce insulin sensitivity and resistance to obesity. Although both rosiglitazone and MRL24 inhibit CDK5 phosphorylation of PPARγ, treatment with the strong agonist rosiglitazone results in upregulation of both the adipogenic and the pro-insulin resistance sets of genes, while treatment with MRL24 results only in upregulation of the pro-insulin resistance set.

The Spiegelman group’s research indicates that the difference between the action of rosiglitazone and MRL24 is due to the different conformational changes induced in the PPARγ protein molecule by binding of the two compounds. The researchers hypothesized that these two different conformational changes may change the way in which PPARγ interacts with coregulator proteins. Nuclear receptors work together with coregulators (i.e., coactivators and corepressors) to regulate specific sets of genes. Different ligands (natural or synthetic) that modulate nuclear receptor interactions with its coregulators can give different results in terms of which genes are unregulated or downregulated.

Researchers hypothesize that it is the upregulation of the adipogenic gene set that is responsible for the adverse effects of strong agonists of PPARγ–weight gain, edema, and the risk of heart failure. In contrast, the upregulation of adiponectin and the other members of the pro-insulin resistance gene set is thought to be responsible for the desirable, antidiabetic effect of PPARγ agonists. If researchers could develop synthetic PPARγ ligands that would induce PPARγ upregulation of the pro-insulin resistance gene set but not the adiopgenic gene set, these compounds might constitute improved, second-generation insulin sensitizers that would have the desirable effects of the TZDs with fewer adverse effects.

In the 22 July 2010 issue of Nature, there are two essays that discuss using the new breakthrough results of the Spiegelman group to discover and develop such improved insulin sensitizers. These are a News and Views article by metabolic disease researchers Riekelt Houtkooper and Johan Auwerx (Ecole Polytechnique Federale de Lausanne [EPFL] in Switzerland), and a Nature News article by Boston-based journalist Heidi Ledford, Ph.D. of Nature.

In the Houtkooper and Auwerx article, the authors advocate changing screening strategies for PPARγ-modulating drugs, to look for those that inhibit CDK5 phosporylation of PPARγ rather than those that are strong PPARγ agonists. Potential PPARγ-modulating drugs would also induce conformational changes in the PPARγ protein that support its recruitment of coregulators that have favorable effects on metabolism (e.g., induce insulin sensitivity and/or protect against obesity). Specifically, these authors suggest that researchers determine whether CDK5-mediated PPARγ phosphorylation enhances recruitment of coactivators such as TIF2/SRC-2 and RIP140 (which unfavorably affect metabolism), and inhibits recruitment of coactivators such as SRC-1 and PGC-1, which have a favorable effect on metabolism. If this is true, drugs that inhibit CDK5 phosporylation of PPARγ should shift the balance toward recruitment of cofactors that favorably affect metabolism (i.e., promote insulin sensitivity and resistance to obesity).

Houtkooper and Auwerx also advocate biochemical and genetic research on the multiple roles of CDK5 and its upstream regulators in metabolic pathways and disorders, as well as research to identify a PPARγ phosphatase that reverses the effects of CDK5 on PPARγ. These authors do not advocate screening for CDK5 inhibitors. (Several already exist, such as roscovitine, which also inhibits several CDKs that are involved in the cell cycle, and is being developed as the anticancer drug Seliciclib by Cyclacel).  CDK5 inhibitors would interfere with CDK5’s other functions, including in the central nervous system. Instead, drug discovery researchers should focus on discovering compounds that specifically change the conformation of PPARγ so that CDK5 phosphorylation of that molecule is inhibited.

In Heidi Ledford’s News article, she also advocates that pharmaceutical companies screen for inhibition of PPARγ phosphorylation, rather than strong activation of PPARγ. She also reviews the rocky history of the “glitazone” class of drugs (i.e., TZDs and related compounds), many of which have exhibited various safety problems that have kept them from the market or removed them from it. (You can see our take on this history in two 2006 articles on our website [here and here). Some commentators therefore believe that companies are unlikely to perform any new screens to identify PPARγ modulators, given the recent conflicts over Avandia and the checkered history of the glitazones. Other commentators counter that because of the large and growing type 2 diabetes market ($20.2 billion in 2008 and a projected $37.9 billion in 2018), companies will be tempted to try some new drug discovery efforts based on the new Spiegelman research. However, others counter that the new Spiegelman results are only preliminary. In any case, if drug developers would start the new screens now, it would take around 10-15 years for any new drugs to appear on the market.

But some companies may not need to start from scratch, by screening for novel compounds that inhibit CDK5 phosphorylation of PPARγ. These companies already have potential second generation insulin sensitizers in clinical development. In our published 2006 article “Safety Issues Hamper Dual PPAR Agonists Is Partial Antagonism the Solution?”, we discussed safety issues both with PPARγ agonists, and with two PPARγ/PPARα dual agonists that were in late-stage development but were discontinued in 2006 because of adverse cardiovascular events. (PPARα is another PPAR nuclear receptor that is involved in control of lipid metabolism, especially of serum triglycerides and high density lipoprotein [HDL].) We proposed that development of what we called “partial agonists” of PPARs might be a solution to these safety problems. “Partial agonists” of PPARγ and of other PPARs are also called “selective PPAR modulators”. In the case of PPARγ, selective modulators are compounds that are less active in activating pathways that result in adipogenesis than strong agonists such as rosiglitzone and pioglitazone, while still upregulating pathways involved in insulin resistance.

In our 2006 article, we especially focused on a compound then called metaglidasen, which was being developed by Metabolex (Hayward, CA) and Johnson & Johnson (JNJ). This non-TZD drug is now called MBX-102/JNJ39659100. Metabolex and JNJ had tested MBX-102 for treatment of type 2 diabetes in eight Phase I and four Phase II clinical studies. However, according to the Metabolex website, the companies have repurposed the drug to treat gout, after discovering that MBX-102 is an effective uricosuric agent (i.e., it increases the excretion of uric acid in the urine).

MBX-102 is a single optical isomer of halofenate, a compound that had been studied in the 1970’s by Merck as a lipid-lowering agent. Halofenate was serendipitously found to be an insulin sensitizer; Metabolex produced a form of the drug that contained only the active optical isomer, and developed it as MBX-102.

in a 2009 paper, researchers showed that MBX-102 had insulin sensitizing and serum glucose-lowering effects in diabetic rat models (but with much lower potency than rosiglitazone), without the weight gain seen with TZDs. In vitro, MBX-102 did not drive human and mouse adipocyte differentiation, unlike TZDs. Moreover, MBX-102 had a greatly reduced ability to recruit PPARγ coactivators. MBX-102’s ability to recruit coactivators that favorably affect metabolism (SRC-1 and PGC-1) was significantly greater than its ability to recruit coactivators that unfavorably affect metabolism (such as TIF2/SRC2). MBX-102 also potently mediated transrepression of proinflammatory genes in vitro and in vivo. Earlier preclinical studies, discussed in our 2006 article, indicated that MBX-102 produced less cardiac hypertrophy in animals than rosglitazone, and also preserved the function of pancreatic beta cells.

Metabolex had also been developing a second selective PPARγ modulator, MBX-2044, a more potent follow-on compound to MBX-102, which reached Phase II of development. However, due to the company’s limited resources, it cannot conduct Phase III clinical trials without partners, especially the large Phase III trials required for diabetes. Therefore, Metabolex has shelved MBX-2044 for now, and has repurposed MBX-102 for gout, in partnership with JNJ. In a June 23 news release, Metabolex characterized itself as “a research-based company…not a commercial company” However, Metabolex is likely to seek to retain co-promotion rights in any commercialization agreement with pharmaceutical companies.

Meanwhile, another biotech company, InteKrin Therapeutics (Los Altos, CA) is developing another selective non-TZD PPARγ modulator, INT131. InteKrin had licensed the drug (formerly known as AMG131 and originally discovered by Tularik) from Amgen in January 2007. Unlike MBX-102, INT131 was purposefully designed to be a selective PPARγ modulator. INT131 has been in Phase II clinical trials for treatment of type 2 diabetes. in a 2009 paper, Amgen researchers showed that INT131 exhibited a different pattern of PPARγ coregulator recruitment from TZDs. in adipocytes, INT131 only minimally activated genes involved in adipogenesis, and exhibited a greater degrees of activation of genes involved in mediating insulin sensitivity. In a diabetic rat model, INT131 had a similar effect on glucose metabolism to rosiglitazone, with similar potency. But unlike rosigliatazone, INT131 did not induce weight gain, cardiac hypertrophy, or edema, in both rodents and nonhuman primates. In Phase IIa clinical trials (as published by InteKrin in 2010, INT131 showed potent glucose lowering (even at the low, 1 mg dose), without weight gain and fluid retention. High-dose (10 mg) INT131 provided apparently greater benefit to glucose metabolism than maximal-dose TZDs, without weight gain and fluid retention. InteKrin has tested INT131 in Phase IIb clinical trials, in which the drug gave comparable glycemic control to pioglitazone, but without edema and with minimal weight gain. The company has completed an End-of-Phase II meeting with the FDA, and is moving INT131 into Phase III clinical trials.

In order to “close the loop” in comparing studies of such compounds as MBX-102, MBX-2044, and INT131 with the new Spiegelman studies of the mechanism of insulin sensitization by PPARγ modulators, researchers would need to test these compounds for inhibition of phosphorylation of PPARγ by CDK5. Such studies have not yet been done.

While we were completing preparation of this three-part series of articles for our blog, a two-page “News and Analysis” article on selective PPAR (including PPARγ and the two other human PPARs) modulators was published in this month’s (September 2010) issue of Nature Reviews Drug Discovery (NRD). As with our blog posts, this article discussed (in a briefer format) the July 2005 FDA Advisory Committee recommendations on Avandia, the recently published Spiegelman article on the effects of insulin sensitizers on PPARγ phosphorylation by CDK5, and the prospects for selective PPARγ modulators as therapeutics for type 2 diabetes.

The NRD article not only discusses selective PPARγ modulators developed by InteKrin and Metabolex (with the latter company’s PPARγ modulators only being listed in a table), but also selective agonists of other PPARs, including single compounds that address multiple PPARs. Among these compounds is Metabolex’ selective PPARδ agonist MBX-8025 (formerly RWJ-800025, in-licensed from Janssen Pharmaceutica). PPARδ regulates genes involved in multiple aspects of the metabolic syndrome, including lipid storage and transport, fatty acid oxidation, and insulin sensitivity. MBX-8025 is being developed for treatment of mixed dyslipidemia and metabolic syndrome. In late 2008, MBX-8025 completed a Phase II proof-of-concept clinical trial, in which the compound was found to substantially reduce serum triglycerides, increase HDL, lower low-density lipoprotein (LDL), and improve insulin sensitivity in obese patients with metabolic syndrome. The effects of MBX-8025 are complementary to the LDL-lowering effects of statins such as atorvastatin (Pfizer’s Lipitor).

Another compound discussed in the NRD article is Roche’s rationally designed balanced dual PPARα/PPARγ activator aleglitazar, which is in Phase III development for treatment of patients with type 2 diabetes who have experienced a recent acute coronary syndrome.  This compound is designed to combine the improvements in insulin sensitivity associated with activation of PPARγ with the amelioration of dyslipidemia associated with activation of PPARα. Roche believes that aleglitazar will avoid the adverse cardiovascular effects seen with earlier dual PPARα/PPARγ activators, or “glitazars”, as discussed in our 2006 article. A 2009 clinical trialshowed that aleglitazar had a positive effect on lipid and glucose metabolism, with no induction of edema or congestive heart failure, and less weight gain than for pioglitazone, over a 16-week treatment period. These short-term results are encouraging, but must be confirmed by the results of ongoing Phase III trials.

Meanwhile, other companies, including Merck, the discoverer of MRL24, have been continuing to develop selective PPARγ modulators for treatment of type 2 diabetes.

In conclusion, the development of selective PPARγ agonists for type 2 diabetes, as we postulated in our 2006 article, is a promising approach to overcoming the issues with current PPARγ agonists, especially rosiglitazone/Avandia. However, it will be important to overcome the cloud that hangs over PPAR modulators, as the result of safety issues with such drug classes as TZDs and glitazars. Encouraging data from ongoing trials of second-generation, selective modulators will hopefully overcome these doubts, enabling companies to develop them and regulators to review them without prejudgment.

It will also be important to “close the loop” with the recent Spiegelman studies, by looking at the effects of these agents on PPARγ phosphorylation by CDK5. Moreover, screening of compounds for their effects on PPARγ phosphorylation may lead to the development of even better agents, especially if agents now in the clinic fail or give less than optimal results in late-stage trials. However, we hope that agents now in development (especially InteKrin’s INT131 and/or the two Metabolex agents if that company can find the resources from partners to complete development for diabetes) will prove to be safe and effective in clinical studies. In the longer term, it will be important to confirm that any new insulin sensitizers work to preserve beta-cell function (which would prevent progression of type 2 diabetes), and to determine if they lower the incidence of cardiovascular complications of diabetes. These are major unmet needs in treatment of type 2 diabetes.

PPARgamma

In part 1 of this three-part series (posted August 24, 2010), we discussed the recent action of the  Endocrinologic and Metabolic Drugs Advisory Committee regarding rosiglitazone (GlaxoSmithKline’s Avandia). The advisory committee, by a close vote, recommended to the FDA that it leave the drug on the market, with new restrictions (e.g., closer supervision and new label warnings).

Avandia and pioglitazone (Takeda’s Actos) are the only marketed members of the thiazolidinedione (TZD) class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists. PPARγ is a nuclear receptor that controls glucose metabolism and adipocyte differentiation. In treatment of type 2 diabetes, TZD modulation of PPARγ results in decreased insulin resistance, thus enabling tissues such as muscle and fat to utilize insulin more efficiently for the uptake of glucose. Agents that work by decreasing insulin resistance are known as “insulin sensitizers”.

As discussed in our August 23 article, clinical evidence indicates that both Avandia and Actos induce weight gain in type 2 diabetics (who are usually obese to begin with), and carry an increased risk of edema and heart failure. Avandia also carries a significantly increased risk of myocardial infarction (MI). Critics of Avandia who want the drug removed from the market cite the increased risk of MI, and the availability of a safer TZD, Actos.

Despite the major safety issues with TZDs, there is both animal model and human evidence that these agents may work to preserve and/or enhance beta-cell function, and thus to help prevent progression of type 2 diabetes. Moreover, insulin resistance is a major factor in the pathogenesis of the disease. We therefore asked whether it might be possible to discover and develop better, safer insulin sensitizers that would have the desirable properties of the TZDs with fewer adverse effects. In this article, which is part 2 of the series, we discuss a recent breakthrough in the biochemistry of PPARγ that may enable companies to develop better insulin sensitizers. In part 3 of this series, we shall look at how companies might develop such compounds.

It was Bruce Spiegelman (Dana-Farber Cancer Institute and Harvard Medical School, Boston MA) and his colleagues who identified PPARγ as the master regulator of adipocyte biology and differentiation back in 1994. This eventually led to the discovery and development of TZDs such as Avandia and Actos, which are synthetic compounds that are strong agonists of PPARγ. These compounds act as potent insulin sensitizers, and are thus used in the treatment of type 2 diabetes. However, the mechanism of their insulin sensitizing activity is not clear. Administration of  TZDs result in decreased expression in adipose tissue of insulin-resistance inducing hormones such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1) and resistin. They also induce increased expression of adiponectin, an adipose-derived hormone or adipokine (a member of a class of cytokines that are secreted by adipocytes) that acts as a natural insulin sensitizer.

In a July 2010 research article published in Nature, the Spiegelman group noted that the action of insulin-sensitizing PPARγ agonists does not make biological sense. Obese people and people with insulin resistance (including type 2 diabetics) have no deficiency in PPARγ or PPARγ activity. Therefore, why do synthetic activators of PPARγ have such dramatic insulin sensitizing and antidiabetic activity? Since PPARγ controls adipocyte differentiation, it makes good biological sense that PPARγ agonists can induce weight gain, however. But why do these inducers of weight gain also act as insulin sensitizers, while excessive weight is generally associated with increased insulin resistance? Finally, although the strong PPARγ agonists rosiglitazone and pioglitazone are insulin sensitizing and antidiabetic, some selective PPARγ modulators with poor agonist activity, such as the (non-TZD) benzoyl 2-methyl indole MRL24 (discovered by Merck Research Laboratories) have very good antidiabetic activity.

The Spiegelman group found that the enzyme cyclin-dependent kinase 5 (CDK5) phosphorylates PPARγ at serine 273 (Ser 273). There are no other CDK5 phosphorylation sites on PPARγ. Unlike other members of the CDK family, CDK5 is not a cell-cycle kinase that is regulated by a cyclin, but instead is regulated by p35/25, which are targets of numerous cytokines and other proinflammatory signals. Specifically, cytoplasmic p35, possibly in response to proinflammatory signals, is cleaved to form p25. p25 can then enter the nucleus, where it associates with and activates CDK5. The activated CDK5 can then phosphorylate PPARγ at Ser 273.

Treatment of adipocytes with proinflammatory cytokines or free fatty acids results in enhanced formation of p25, and enhanced phosphorylation of PPARγ at Ser 273. The same results occur in vivo, in mice that are fed a high-fat diet over a prolonged period of time, and thus become obese. It is well known that both free fatty acids and proinflammatory cytokines are elevated in obesity.

The researchers also found that phosphorylation of PPARγ at Ser 273 does not change the ability of PPARγ to upregulate transcription of genes involved in adipocyte differentiation. However, it inhibits the ability of PPARγ to upregulate transcription of certain other genes, including adiponectin. The insulin-sensitizing synthetic compounds rosiglitazone (a strong PPARγ agonist) and MRL24 (a weak PPARγ agonist) both inhibited phosphorylation of PPARγ at Ser 273 in adipocytes. However, treatment of adipocytes with these two compounds gave different results in terms of their effects on PPARγ-regulated genes. Treatment of fat cells with rosiglitazone resulted in upregulation of adiponectin and other genes that are downregulated by Ser 273 phosphorylation. But rosiglitazone treatment also resulted in upregualtion of PPARγ-regulated genes involved in adipogenesis. In contrast, treatment of adipocytes with MRL24 did not upregulate the genes involved in adipogenesis. But it did upregulate the gene set (including adiponectin) that was downregulated by phosphorylation of PPARγ at Ser 273.

Mass spectrometry studies indicated that both rosiglitazone and MRL24 changed the conformation of PPARγ in such a way as to make this protein less favorable for phosphorylation by CDK5. However, rosiglitazone and MRL24 binding results in different conformational changes. The researchers hypothesized that these conformational changes may change the way in which PPARγ interacts with coregulator proteins. Nuclear receptors work together with coregulators to regulate specific sets of genes. Different ligands (natural or synthetic) that modulate nuclear receptor interactions with its coregulators can give different results in terms of which genes are unregulated or downregulated.

The researchers then studied the action of roslglitazone and of MRL24 on phosphorylation of PPARγ at Ser 273 and on modulation of PPARγ-regulated genes in vivo. In mouse models, these two compounds inhibited PPARγ Ser 273 phosphorylation in adipose tissue, and caused similar changes in PPARγ-regulated gene expression as they do in adipose cells in vitro. Moreover, human diabetes patients treated with rosiglitazone usually exhibited decreased phosphorylation of PPARγ at Ser 273 in biopsied subcutaneous fat. However, in some cases, no such decreased phosphorylation was seen. Improvements in insulin sensitivity in these patients correlated with decreased phosphorylation of PPARγ at Ser 273.

On the basis of these results, the researchers concluded that the insulin sensitizing and antidiabetic effects of PPARγ agonists may not be due to the agonistic effects of these compounds on PPARγ, but on their ability to inhibit CDK5 phosphorylation of PPARγ.

In a News and Views article in the same issue of Nature, Riekelt Houtkooper and Johan Auwerx (Ecole Polytechnique Federale de Lausanne [EPFL] in Switzerland) postulate that the new findings of the Spiegelman group may be used to develop PPARγ modulating drugs that lack full agonist activity, but still inhibit CDK5 phosphorylation of PPARγ. Such compounds (of which MRL24 may be a starting point) would not upregulate adipogenic genes, but would upregulate insulin sensitizing genes such as adiponectin. These authors postulate that these novel compounds may thus have strong insulin sensitizing and antidiabetic effects, but would lack such adverse effects as weight gain, edema, and the risk of heart failure.

We shall discuss strategies for developing improved insulin sensitizers in greater depth in Part 3 of this series.

B-Raf

In March 2010, we published two articles on this blog relating to Roche/Plexxikon’s PLX4032 for metastatic melanoma. The first article, dated March 2, described a Phase I clinical trial of the drug, based on an article about this trial in the New York Times (NYT). The second article, dated March 10, described Plexxikon’s discovery of PLX4032, using its proprietary “scaffold-based drug design” technology platform. The latter post is among the most popular articles on this blog.

Now the results of the Phase I trial of PLX4032 has been published in the August 26, 2010 issue of the New England Journal of Medicine (NEJM). (A subscription is required to read the full article.)

As we discussed in our previous articles, PLX4032 is a B-Raf (called “BRAF” in the NEJM paper and in some other publications) kinase inhibitor that is exquisitely specific for B-Raf carrying the V600E mutation. B-Raf(V600E) is the most common somatic mutation found in human melanomas. Researchers believe that B-Raf(V600E) is a “driver mutation” that is particularly critical for the malignant phenotype of human metastatic melanomas that carry the mutation. B-Raf(V600E) is constitutively activated, and melanomas carrying this mutation can proliferate independently of growth factor signaling, resulting in the runaway proliferation characteristic of the malignant phenotype.

The clinical trial described in the NEJM article was carried out by researchers at Plexxikon and Roche, in collaboration with academic researchers at five institutions in the United States and Australia. The trial was led by Keith T. Flaherty, M.D. (then at the University of Pennsylvania in Philadelphia, and now at the Massachusetts General Hospital Cancer Center [where he is Director of Developmental Therapeutics] and the Dana-Farber Cancer Institute in Boston) and Paul B. Chapman, MD (Memorial Sloan-Kettering Cancer Center).

As discussed in the NEJM article, the researchers conducted a multicenter Phase I dose-escalation trial of PLX4032 (which is orally available), followed by an extension phase in which patients were given the maximum dose that could be administered without adverse effects (960 mg twice daily). (The latter dose is the recommended Phase II dose.) A total of 55 patients (49 of whom had melanoma) were enrolled in the initial, dose-escalation portion of the trial. 32 additional patients, all of whom had metastatic melanoma with the B-Raf(V600E) mutation, were enrolled in the extension phase. Patients were given the drug twice a day until they had disease progression.

In the dose-escalation phase, among the 16 patients with melanoma carrying the B-Raf(V600E) mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response (i.e., tumor shrinkage of at least 30%) and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The latter figure represents an 81% response rate. The estimated median progression-free survival among all patients was over 7 months.

Dose-limiting adverse effects included rash, fatigue, and joint pain.

The published results of this Phase I trial elicited great enthusiasm in the popular press and in such industry media as Fierce Biotech and BioWorld Online, and by oncologists who were interviewed for these articles. The oncologists said that they had never seen such a dramatic response in treatment of metastatic melanoma.

Because PLX4032 is targeted to a specific oncogenic mutation, Plexxikon and several industry commentators refer to the use of the drug as an example of personalized medicine. In parallel with development of PLX4032, Plexxikon and Roche Molecular Systems are developing a DNA-based companion diagnostic to identify patients whose tumors carry the B-Raf(V600E) mutation.

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

Meanwhile, the results of a Phase III trial (in 676 patients with advanced melanoma) of Medarex/Bristol-Myers Squibb’s (BMS’s) ipilimumab were published in the August 19, 2010 issue of the NEJM.  Ipilimumab, unlike the targeted therapeutic PLX4032, is an immunomodulator that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) to potentate an antitumor T-cell response. Ipilimumab is a monoclonal antibody, unlike PLX4032 which is a small-molecule compound. In this NEJM article, the researchers reported that ipilimumab–given with or without the 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. BMS has filed a Biologics License Application (BLA) for ipilimumab, and earlier this month (August 2010) received fast-track status from the FDA for the drug.

Ipilimumab treatment is associated with autoimmune toxicities (especially enterocolitis), which can be severe. These are usually reversible by treatment with high-dose steroids.

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.

We believe that it is important to pursue development of both targeted therapies and of immunomodulators for metastatic melanoma. This may provide oncologists a range of therapeutics (and of combinations of therapeutics) to treat this disease, which now has very few treatment options and a very poor prognosis.

The results with both PLX4032 and ipilimumab provide hope for better treatment of at least some classes of metastatic melanoma in the near future. However, as discussed in our March 2010 articles, even in the case of PLX4032 treatment of melanoma carrying the B-Raf(V600E) mutation, it will most likely be necessary to develop combination therapies in order to achieve long-lasting remissions or cures.

Alpha-helix

On November 27, 2009, we posted an article on this blog about the use of stapled peptides in targeting intracellular pathways. This technology was originally developed by Dr. Gregory Verdine (Department of Chemical Biology, Harvard University, Cambridge MA, and the Dana-Farber Cancer Institute, Boston MA) and his colleagues. A biotechnology company, Aileron Therapeutics (Cambridge, MA) was founded (with Dr. Verdine among its founders) in 2005 to develop and commercialize stapled peptide drugs. Aileron’s most advanced compounds, which are being developed for the treatment of solid and hematological tumors, are only in the preclinical stage.

On August 24, 2010, Aileron and Roche announced that they had entered into a collaboration to discover, develop, and commercialize stapled peptide drugs designed to address up to five undisclosed targets. These targets are selected from Roche’s key therapeutic areas of interest–oncology, viral diseases, inflammation, metabolic diseases, and central nervous system diseases.

Under the agreement, Roche will provide Aileron guaranteed funding of at least $25 million in R&D support and technology access fees. Aileron will also be eligible to receive up to $1.1 billion in discovery, development, regulatory, and commercialization milestone payments, if drug candidates are developed against all five targets. Aileron will also receive royalties on any future sales of marketed products that result from the collaboration.

In our November 2009 article, we discussed the design of stapled peptides, in which hydrocarbon moieties are used to constrain, or “staple” peptide sequences into an α-helical conformation. These sequences are designed to mimic key binding domains of proteins that are involved in intracellular signaling pathways. We gave two examples of pathways that were addressed by specific stapled peptides: the Notch pathway and a Bcl-2-related apoptotic pathway. In both cases, the stapled peptides modulated protein-protein interactions that are considered “undruggable” by conventional small-molecule drugs.

According to Roche, It is “as yet intractable” intracellular protein-protein interactions that are of special interest to the company in collaborating with Aileron.

According to Aileron, the new alliance with Roche validates the broad potential of their stapled peptide technology platform across multiple therapeutic areas and classes of targets. The alliance also provides Aileron with capital to advance its internal R&D.

The Roche agreement represents Aileron’s first Big Pharma strategic alliance. However, a venture capital consortium that included GlaxoSmithKline, Novartis, Roche, and Lilly invested $40 million in Aileron in June 2009.

As we said in our November 2009 article, stapled peptides represent an exciting and innovative technology with the potential to address “undruggable” protein-protein interactions, even though the therapeutic value of stapled peptides has not yet been confirmed in the clinic. (We have discussed several other means of addressing protein-protein interactions in various articles in this blog–these targets represent an area of opportunity for companies that are innovative enough to pursue it.) And as we discussed in a more recent article, Roche is one of the Big Pharma companies that continues to be focused on innovative drug discovery and development, in an era of Big Pharma R&D retrenchment. The Aileron-Roche partnership therefore appears to be an ideal match.

Avandia (rosiglitazone)

On July 15, 2010, the FDA’s Endocrinologic and Metabolic Drugs Advisory Committee voted to leave the diabetes drug rosiglitazone  (GlaxoSmithKline’s  Avandia) on the market, with some new restrictions (e.g., closer supervision and new label warnings). This is the same committee that voted against FDA approval of Vivus’ anti-obesity drug Qnexa on the same day, as discussed in our August 4 blog post. (Some commentators believe that the Qnexa rejection is connected to the decision on Avandia. We shall reserve judgment on that question.)

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

Of the 33-member panel, 10 voted to keep Avandia on the market under close supervision, seven voted to keep it on the market but with stronger label warnings, and three voted to keep the drug on the market with no new restrictions. Twelve voted to remove Avandia from the market. One member abstained.

One factor in the recommendations of several panelists to add restrictions or to eliminate Avanida from the market altogether is that a competing drug of the same thiazolidinedione (TZD) class, pioglitazone (Takeda’s Actos), appears to have similar efficacy to Avandia, but fewer adverse effects.

The panel members’ decisions were based on their analysis of large amounts (18 presentations worth) of contradictory data.

TZDs are agonists of peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor that controls glucose metabolism and adipocyte differentiation. In treatment of type 2 diabetes, TZD modulation of PPARγ results in decreased insulin resistance. (Insulin resistance is the inability of tissues such as muscle and fat to utilize insulin efficiently for the uptake of glucose.) Thus treatment with these drugs can result in decreased levels of serum glucose, and amelioration of diabetes. Agents that work by decreasing insulin resistance are known as “insulin sensitizers”.

We have been following safety issues with agonists of PPAR receptors for quite some time. For example, there are two articles (here and here) published in 2006 (and available on our website) that include discussion of PPAR agonist safety.

As discussed in these two articles, Steven Nissen, M.D. (now Chairman of the Robert and Suzanne Tomsich Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH) has been a leading critic of Avandia’s cardiac safety. This began with his 2007 meta-analysis of clinical studies of the drug, published in the New England Journal of Medicine. This meta-analysis indicated that treatment with Avandia was associated with a significant increase in the risk of myocardial infarction (MI), and an increase in the risk of cardiovascular death that had borderline significance. A 2010 meta-analysis by Dr. Nissen and his colleagues (published online ahead of print in the Archives of Internal Medicine on June 28) indicated that treatment with Avandia was associated with a significant increase in the risk of MI, but no increased risk of cardiovascular death or all-cause mortality. The results also suggest an unfavorable benefit to risk ratio for Avandia.

A retrospective study by FDA researchers of Medicare recipients published in the Journal of the American Medical Association in July 2010 indicated that Avandia treatment is associated with an increased risk of the composite of AMI, stroke, heart failure, and all-cause mortality as compared with Actos, in patients 65 years or older.

The consensus of multiple studies is that both Avandia and Actos induce weight gain, and carry an increased risk of edema and heart failure as compared with placebo, but that Avandia has a higher risk of MI than Actos. (Few studies directly compared the two drugs, however. The 2010 FDA study is an exception). However, some studies presented to the FDA Advisory Committee indicated that the risk of cardiovascular events were comparable between Avandia, Actos, and diabetes medications of other classes.

The panel’s decision was a compromise, based not only on the contradictory nature of the evidence, but also on the contention that Avandia may be a better choice than Actos (or other diabetes drugs) for certain groups of patients, but not others. However, the continuing bad publicity about Avandia’s risks have significantly reduced its sales.

The continuing unfavorable safety findings for Avandia, as well as the findings that both Avandia and Actos induce weight gain in type 2 diabetics (who are usually obese to begin with), and carry an increased risk of edema and heart failure, have given new ammunition to critics who believe that physicians treating diabetes should stick to combinations of the older, cheaper drugs–insulin, metformin, and sulfonylureas, and avoid using not only TZDs, but also newer agents. This point of view also suggests that there is no need to discover and develop new antidiabetic agents.

However, the arguments in our 2008 Genetic Engineering News (GEN) article on diabetes (available on our website) still apply. There are still major unmet needs in type 2 diabetes, especially the need to prevent weight gain in diabetes treatment (and even to promote weight loss), and the need to prevent long-term loss of pancreatic beta-cell function. It is the loss of beta-cell function that results in the progression of type 2 diabetes, such that patients who initially succeed in reaching glycemic goals even with multidrug treatment with older antidiabetics eventually experience poor glycemic control on the same regimens.

As we discussed in earlier blog posts, some of the newer antidiabetics, namely the incretin mimetic exenatide (Amylin/Lilly’s Byetta) and liraglutide (Novo Nordisk’s Victoza), may give increased glycemic control while promoting weight loss. There is also evidence from animal studies that these drugs might help to preserve beta-cell function.

Ironically, despite the major safety issues with TZDs, there is both animal model and human evidence that these agents may work to preserve and/or enhance beta-cell function. Moreover, insulin resistance is a major factor in the pathogenesis of type 2 diabetes. Therefore, it would be very advantageous, and perhaps essential, for physicians and patients to have access to safer insulin sensitizers, especially if they work to prevent diabetes progression by preserving and enhancing beta-cell function.

Might it be possible to discover and develop better, safer insulin sensitizers than the TZDs? We shall discuss this question in Part 2 and Part 3 of this series.