CXCR-1 N-terminal peptide bound to IL-8

In our October 31, 2013 blog article, we discussed recent structural studies of the chemokine receptors CCR5 and CXCR4. We discussed the implications of these studies for the treatment of HIV/AIDS, especially using the CCR5 inhibitor maraviroc (Pfizer’s Selzentry/Celsentri). As discussed in the article, researchers are utilizing the structural studies of CCR5 and CXCR4 to develop improved HIV entry inhibitors that target these chemokine receptors.

Meanwhile, other researchers have been studying the role of chemokine receptors in cancer biology, and the potential use of chemokine receptor antagonists in cancer treatment.

CCR5 antagonists as potential treatments for metastatic breast cancer

One group of researchers, led by Richard G. Pestell, M.D., Ph.D. (Thomas Jefferson University, Philadelphia, PA) has been studying expression of CCR5 and its ligand CCL5 (also known as RANTES) and their role in breast cancer biology and pathogenesis. Their report of this study was published in the August 1, 2012 issue of Cancer Research.

These researchers first studied the combined expression of CCL5 and CCR5 in various subtypes of breast cancer, by analyzing a microarray database of over 2,000 human breast cancer samples. (The database was compiled from 27 independent studies). They found that CCL5/CCR5 expression was preferentially expressed in the basal and HER-2 positive subpopulations of human breast cancer.

Because of the high level of unmet medical need in treatment of basal breast cancer, the authors chose to focus their study on this breast cancer subtype. As the researchers point out, patients with basal breast cancer have increased risk of metastasis and low survival rates. Basal tumors in most cases do not express either androgen receptors, estrogen receptors (ERs), or HER-2. They thus cannot be treated with such standard receptor-targeting breast cancer therapeutics as tamoxifen, aromatase inhibitors, or trastuzumab. The only treatment options are cytotoxic chemotherapy, radiation, and/or surgery. However, these treatments typically results in early relapse and metastasis.

The basal breast cancer subpopulation shows a high degree of overlap with triple-negative (TN) breast cancer. We discussed TN breast cancer, and research aimed at defining subtypes and driver signaling pathways, in our August 2, 2011 article on this blog. In that article, we noted that TN breast cancers include two basal-like subtypes, at least according to one study. Other researchers found that 71% of TN breast cancers are of basal-like subtype, and that 77% of basal-like tumors are TN. A good part of the problem is that there is no accepted definition of basal-like breast cancers, and how best to define such tumors is controversial. However, both the TN and the basal subpopulations are very difficult to treat and have poor prognoses. It is thus crucial to find novel treatment strategies for these subpopulations of breast cancer.

Dr. Pestell and his colleagues therefore investigated the role of CCL5/CCR5 signaling in three human basal breast cancer cell lines that express CCR5. They found that CCL5 promoted intracellular calcium (Ca2+) signaling in these cells. The researchers then determined the effects of CCL5/CCR5 signaling in promoting in vitro cell invasion in a 3-dimensional invasion assay. For this assay, the researchers assessed the ability of cells to move from the bottom well of a Transwell chamber, across a membrane and through a collagen plug, in response to CCL5 as a chemoattractant. The researchers found that CCR5-positive cells, but not CCR5-negative cells, showed CCL5-dependent invasion.

The researchers then studied the ability of CCR5 inhibitors to block calcium signaling and in vitro invasion. The agents that they investigated were maraviroc and vicriviroc. Maraviroc (Pfizer’s Selzentry/Celsentri) is the marketed HIV-1 entry inhibitor that we discussed in our October 31, 2013 article.  Vicriviroc is an experimental HIV-1 inhibitor originally developed by Schering-Plough. Schering-Plough was acquired by Merck in 2009. Merck discontinued development of vicriviroc because the drug failed to meet primary efficacy endpoints in late stage trials.

Pestell et al. found that maraviroc and vicriviroc inhibited calcium responses by 65% and 90%, respectively in one of their CCR5-positive basal cell breast cancer lines, and gave similar results in another cell line. The researchers then found that  in two different CCR5-positive basal breast cancer cell lines, both maraviroc and vicriviroc inhibited in vitro invasion.

The researchers then studied the effect of maraviroc in blocking in vivo metastasis of a CCR5-positive basal cell breast cancer line, which had been genetically labeled with a fluorescent marker to facilitate noninvasive visualization by in vivo bioluminescence imaging (BLI). They used a standard in vivo lung metastasis assay, in which cells were injected into the tail veins of immunodeficient mice, and mice were treated by oral administration with either maraviroc or vehicle. The researchers then looked for lung metastases. They found that maraviroc-treated mice showed a significant reduction in both the number and the size of lung metastases, as compared to vehicle-treated mice.

In both in vitro and in vivo studies, the researchers showed that maraviroc did not affect cell viability or proliferation. In mice with established lung metastases, maraviroc did not affect tumor growth. Maraviroc inhibits only metastasis and homing of CCR5-positive basal cell breast cancer cells, but not their viability or proliferation.

As the result of their study, the researchers propose that CCR5 antagonists such as maraviroc and vicriviroc may be useful as adjuvant antimetastatic therapies for breast basal tumors with CCR5 overexpression.  They may also be useful as adjuvant antimetastatic treatments for other tumor types where CCR5 promotes metastasis, such as prostate and gastric cancer.

As usual, it must be emphasized that although this study is promising, it is only a preclinical proof-of-principle study in mice, which must be confirmed by human clinical trials.

In an October 25, 2013 Reuters news story, it was revealed that Citi analysts believe that Merck will take vicriviroc into the clinic  in cancer patients in 2014. Citi said that it expected vicriviroc to be tested in combination with “a Merck cancer immunotherapy” across multiple cancer types, including melanoma, colorectal, breast, prostate and liver cancer. (We discussed Merck’s promising cancer immunotherapy agent lambrolizumab/MK-3475 in our June 25, 2013 blog article. But the Merck agent to be tested together with vicriviroc was not disclosed in the Reuters news story.)

Despite this news story, Merck said that it had not disclosed any plans for clinical trials of vicriviroc in cancer.

The CXCR1 antagonist reparixin as a potential treatment for breast cancer

In our In April 2012 book-length report, “Advances in the Discovery of Protein-Protein Interaction Modulators” (published by Informa’s Scrip Insights), we discussed the case of the allosteric chemokine receptor antagonist reparixin (formerly known as repertaxin). Reparixin has been under developed by Dompé Farmaceutici (Milan, Italy). This agent targets both CXCR1 and CXCR2, which are receptors for interleukin-8 (IL-8). IL-8 is a well-known proinflammatory chemokine that is a major mediator of inflammation. As we discussed in our report, reparixin had been in Phase 2 development for the prevention of primary graft dysfunction after lung and kidney transplantation. However, it failed in clinical trials.

Meanwhile, researchers at the University of Michigan (led by Max S. Wicha, M.D., the Director of the University of Michigan Comprehensive Cancer Center) and at the Institut National de la Santé et de la Recherche Médicale (INSERM) in France were working to define a breast cancer stem cell signature using gene expression profiling. They found that CXCR1 was among the genes almost exclusively expressed in breast cancer stem cells, as compared with its expression in the bulk tumor.

IL-8 promoted invasion by the cancer stem cells, as demonstrated in an in vitro invasion assay. The CXCR1-positive, IL-8 sensitive cancer stem cell population was also found to give rise to many more metastases in mice than non-stem cell breast tumor cells isolate from the same cell line. This suggested the hypothesis that a CXCR1 inhibitor such as reparixin might be used as an anti-stem cell, antimetastatic agent in the treatment of breast cancer.

Dr. Wicha and his colleagues then studied the effects of blockade of CXCR1 by either reparixin or a CXCR1-specific blocking antibody on  bulk tumor and cancer stem cells in two breast cancer cell lines. The researchers found in in vitro studies that treatment with either of these two CXCR1 antagonists selectively depleted the cell lines of cancer stem cells (which represented 2% of the tumor cell population in both cell lines).

This depletion was followed by the induction of massive apoptosis of the bulk, non-stem tumor cells. This was mediated via a bystander effect, in which CXCR1-inhibited stem cells produce the soluble death mediator FASL (FAS ligand). FASL binds to FAS receptors on the bulk tumor cells, and induces an apoptotic pathway in these cells that results in their death.

In in vivo breast cancer xenograft models, the researchers treated tumor-bearing mice with either the cytotoxic agent docetaxel, reparixin, or a combination of both agents. Docetaxel treatment–with or without reparixin–resulted in a significant inhibition of tumor growth, while reparixin alone gave only a modest reduction in tumor growth. However, treatment with docetaxel alone gave no reduction (or an increase) in the percentage of stem cells in the tumors, while reparixin–either alone or in combination with docetaxel–gave a 75% reduction in the percentage of cancer stem cells. Moreover, in in vivo metastasis studies in mice, reparixin treatment gave a major reduction in systemic metastases. These results suggest that reparixin may be useful in eliminating breast cancer stem cells and in inhibiting metastasis and thus preventing recurrence of cancer in patients treated with chemotherapy.

As we discussed in our 2012 report, Dr. Wicha’s research on reperixin might represent an opportunity for Dompé to repurpose reperixin for cancer treatment. Since the publication of the 2012 report, Dompé has been carrying out a Phase 2 pilot study of reparixin in patients diagnosed with early, operable breast cancer, prior to their treatment via surgery. The goal of this study is to investigate if cancer stem cells decrease in two early breast cancer subgroups (estrogen receptor-positive and/or progesterone receptor positive/HER-2-negative, and estrogen receptor negative/progesterone receptor negative/HER-2-negative). The goal is to compare any differences between the two subgroups in order to better identify a target population.

Dompé has thus begun the process of clinical evaluation of reparixin for the new indication–treatment of breast cancer in order to inhibit metastasis and prevent recurrence.

Conclusions

Researchers have found promising evidence that at least two chemokine/chemokine receptor combinations may be involved in cancer stem cell biology and thus in the processes of metastasis and cancer recurrence. In at least one case–and perhaps both–companies are in the early stages of developing small-molecule chemokine receptor antagonists for inhibiting breast cancer metastasis and recurrence. Such a strategy might be applicable to other types of cancer as well.

As discussed by Wicha et al., in immune and inflammatory processes, chemokines serve to facilitate the homing and migration of immune cells. In the case of cancer, chemokines may act as “stemokines”, by facilitating the homing of cancer stem cells in the process of metastasis. Other chemokines and their receptors than those discussed in this article may be involved in other types of cancer, and may carry out similar “stemokine” functions.

Since around 90% of cancer deaths are due to metastasis, and since effective treatments for metastatic cancers are few, this is a potentially important area of cancer research and drug development.

As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company,  please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Maraviroc

In April 2012, Informa’s Scrip Insights published our book-length report, “Advances in the Discovery of Protein-Protein Interaction Modulators.” We also published a brief introduction to this report, highlighting the strategic importance of protein-protein interaction (PPI) modulators for the pharmaceutical industry, on the Biopharmconsortium Blog.

The report included a discussion on discovery and development of inhibitors of chemokine receptors. Chemokine receptors are members of the G-protein coupled receptor (GPCR) superfamily. GPCRs are seven-transmembrane (7TM) domain receptors (i.e. integral membrane proteins that have seven membrane-spanning domains). Compounds that target GPCRs represent the largest class of drugs produced by the pharmaceutical industry. However, in the vast majority of cases, these compounds target GPCRs that bind to natural small-molecule ligands.

Chemokine receptors, however, bind to small proteins, the chemokines. These proteins constitute a class of small cytokines that guide the migration of immune cells via chemotaxis. Chemokine receptors are thus a class of GPCRs that function by forming PPIs. Direct targeting of interactions between chemokines and their receptors (unlike targeting the interactions between small-molecule GPCR ligands and their receptors) thus involves all the difficulties of targeting other types of PPIs.

However, GPCRs–including chemokine receptors–appear to be especially susceptible to targeting via allosteric modulators. Allosteric sites lie outside the binding site for the protein’s natural ligand. However, modulators that bind to allosteric sites change the conformation of the protein in such a way that it affects the activity of the ligand binding site. (Direct GPCR modulators that bind to the same site as the GPCR’s natural ligands are known as orthosteric modulators.) In the case of chemokine receptors, researchers can in some cases discover small-molecule allosteric modulators that activate or inhibit binding of the receptor to its natural ligands. Discovery of such allosteric activators is much easier than discovery of direct PPI modulators.

Chemokines bind to sites that are located in the extracellular domains of their receptors. Allosteric sites on chemokine receptors, however, are typically located in transmembrane domains that are distinct from the chemokine binding sites. Small-molecule allosteric modulators that bind to these sites were discovered via fairly standard medicinal chemistry and high-throughput screening, sometimes augmented with structure-based drug design. This is in contrast to attempts to discover small molecule agents that directly inhibit binding of a chemokine to its receptor, which has so far been extremely challenging.

Our report describes several allosteric chemokine receptor modulators that are in clinical development, as well as the two agents that have reached the market. One of the marketed agents, plerixafor (AMD3100) (Genzyme’s Mozobil), is an inhibitor of the chemokine receptor CXCR4. It is used in combination with granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells to the peripheral blood for autologous transplantation in patients with non-Hodgkin lymphoma and multiple myeloma. The other agent, which is the focus of this blog post, is maraviroc (Pfizer’s Selzentry/Celsentri).

Maraviroc is a human immunodeficiency virus-1 (HIV-1) entry inhibitor. This compound is an antagonist of the CCR5 chemokine receptor. CCR5 is specific for the chemokines RANTES (Regulated on Activation, Normal T Expressed and Secreted) and macrophage inflammatory protein (MIP) 1α and 1β.  In addition to being bound and activated by these chemokines, CCR5 is a coreceptor (together with CD4) for entry of the most common strain of HIV-1 into T cells. Thus maraviroc acts as an HIV entry inhibitor; this is the drug’s approved indication in the U.S. and in Europe. Maraviroc was discovered via a combination of high-throughput screening and optimization via standard medicinal chemistry.

New structural biology studies of the CCR5-maraviroc complex

Now comes a report in the 20 September 2013 issue of Science on the structure of the CCR5-maraviroc complex. This report was authored by a mainly Chinese group led by Beili Wu, Ph.D. (Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai); researchers at the University of California at San Diego and the Scripps Research Institute, San Diego were also included in this collaboration. A companion Perspective in the same issue of Science was authored by P. J. Klasse, M.D., Ph.D. (Weill Cornell Medical College, Cornell University, New York, NY).

As described in the Perspective, the outer surface of the HIV-1 virus displays numerous envelope protein (Env) trimers, each including the outer gp120 subunit anchored in the viral membrane by gp41. When gp120 binds to the cell-surface receptor CD4, this enables interaction with a specific chemokine receptor, either CCR5 or CXCR4. Interaction with both CD4 and the chemokine receptor triggers complex sets of changes in the Env complex, eventually resulting in the fusion of the viral membrane and the cell membrane, and the entry of the virus particle into the host cell.

HIV-1 gp120 makes contact with CCR5 at several points. The interactions between CCR5 and the variable region of gp120 called V3 are especially important for the tropism of an HIV-1 strain, i.e., whether the virus is specific for CCR5 (the “R5 phenotype”) or CXCR4 (the “X4 phenotype”). In the case of R5-tropic viruses, the tip of the V3 region interacts with the second extracellular loop (ECL2) of CCR5, while the base of V3 interacts with the amino-terminal segment of CCR5. Modeling of the interactions between the V3 domain of gp120 of either R5 or X4-tropic viruses with CCR5 or CXCR4 explains coreceptor use, in terms of forming strong bonds or–conversely–weak bonds and steric hindrance.

Monogram Biosciences (South San Francisco, CA) has developed and markets the Trofile assay. This is a molecular assay designed to identify the R5, X4, or mixed tropism of a patient’s HIV strain. If a patient’s strain is R5-tropic, then treatment with maraviroc is appropriate. However, a patient’s HIV-1 strain may undergo a tropism switch, or may mutate in other ways to become resistant to maraviroc.

Dr Wu and her colleagues determined the high-resolution crystal structure of the complex between maraviroc and a solubilized engineered form of CCR5. This included determining the CCR5 binding pocket for maraviroc, which was determined both by Wu et al’s X-ray crystallography, and by site-directed mutagenesis (i.e., to determine amino acid residues that are critical for maraviroc binding) that had been published earlier by other researchers.

The structural studies of Dr. Wu and her colleagues show that the maraviroc-binding site is different from the recognition sites for gp120 and for chemokines, as expected for an allosteric inhibitor. The X-ray structure shows that maraviroc binding prevents the helix movements that are necessary for binding of g120 to induce the complex sequence of changes that result in fusion between the viral and cellular membranes. (These helix movements are also necessary for induction of signal transduction by binding of chemokines to CCR5.)

Structural studies of CXCR4 and its inhibitor binding sites

In addition to their structural studies of the CCR5-maraviroc complex, Dr. Wu and her colleagues also published structural studies of CXCR4 complexed with small-molecule and cyclic peptide inhibitors in Science in 2010. These inhibitors are IT1t, a drug-like orally-available isothiourea developed by Novartis, and CVX15, a 16-residue cyclic peptide that had been previously characterized as an HIV-inhibiting agent. IT1t and CVX15 bind to overlapping sites in CXCR4. Other researchers have found evidence that the binding site for plerixafor also overlaps with the IT1t binding site.

As discussed in Wu et al’s 2013 paper, CCR5 and CXCR4 have similar, but non-identical structures. The binding site for IT1t in CXCR4 is closer to the extracellular surface than is the maraviroc binding site in CCR5, which is deep within the CCR5 molecule. The entrance to the CXCR4 ligand-binding pocket is partially covered by CXC4’s N terminus and ECL2, but the CCR5 ligand-binding pocket is more open.

Mechanisms of CXCR4 and CCR5 inhibition, and implications for discovery of improved HIV entry inhibitors

The chemokine that specifically interacts with the CXCR4 receptor is known as CXCL12 or stromal cell-derived factor 1 (SDF-1). Researchers have proposed a hypothesis for how CXCL12 interacts with CXCR4; this hypothesis appears to be applicable to the interaction between other chemokines and their receptors as well. This hypothesis is know as the “two-step model” or the “two-site model” of chemokine-receptor activation. Under the two-site model, the core domain of a chemokine binds to a site on its receptor (known as the “chemokine recognition site 1” or “site 1”) defined by the receptor’s N-terminus and its ECLs. In the second step, the flexible N-terminus of the chemokine interacts with a second site (known as “chemokine recognition site 2” or “site 2” or the “activation domain”) deeper within the receptor, in transmembrane domains. This result in activation of the chemokine receptor and intracellular signaling.

Under the two-site model, CXCR4 inhibitors (e.g., IT1t, CVX15, and  plerixafor), which bind to sites within the ECLs of CXCR4, are competitive inhibitors of binding of the core domain of CXCL12 to CXCR4 (i.e.., step 1 of chemokine/receptor interaction). They are thus orthosteric inhibitors of CXCR4. (This is contrary to the earlier assignment of plerixafor as an allosteric inhibitor of CXCR4.)  The CCR5 ligand maraviroc, however, binds within a site within the transmembrane domains of CCR5, which overlaps with the activation domain of CCR5. Dr. Wu and her colleagues propose two alternative hypotheses: 1. Maraviroc may inhibit CCR5 activation by chemokines by blocking the second step of chemokine/chemokine receptor interaction, i.e., receptor activation. 2. Maraviroc may stabilize CCR5 in an inactive conformation. It is also possible that maraviroc inhibition of CCR5 may work via both mechanisms.

Dr. Wu and her colleagues further hypothesize that the interaction of  HIV-1 gp120 with CCR5 (or CXCR4) may operate via similar mechanisms to the interaction of chemokines with their receptors. As we discussed earlier in this article, the base (or the stem region) of the gp120 V3 domain interacts with the amino-terminal segment of CCR5. The tip (or crown) of the V3 domain interacts with the ECL2 of CCR5, and–according to Dr. Wu and her colleagues–also with amino acid residues inside the ligand binding pocket; i.e., the activation site of CCR5. The HIV gp120 V3 domain may thus activate CCR5 via a similar mechanism to the two-step  model utilized by chemokines.

Based on their structural biology studies, Dr. Wu and her colleagues have been building models of the CCR5-R5-V3 and CXC4-X4-V3 complexes, and are also planning to determine additional structures needed to fully understand the mechanisms of HIV-1 tropism. The researchers will utilize their studies in the discovery of improved, second-generation HIV entry inhibitors for both R5-tropic and X4-tropic strains of HIV-1.

The bigger picture

The 17 October 2013 issue of Nature contains a Supplement entitled “Chemistry Masterclass”. In that Supplement is an Outlook review entitled “Structure-led design”, by Nature Publishing Group Senior Editor Monica Hoyos Flight, Ph.D. The subject of this article is structure-based drug design of modulators of GPCRs.
This review outlines progress in determining GPCR structures, and in using this information for discovery of orthosteric and allosteric modulators of GPCRs.

According to the article, the number of solved GPCR structures has been increasing since 2008, largely due to the efforts of the Scripps GPCR Network, which was established in that year. Dr. Wu started her research on CXCR4 and CCR5 as a postdoctoral researcher in the laboratory of Raymond C. Stevens, Ph.D. at Scripps in 2007, and continues to be a member of the network. The network is a collaboration that involves over a dozen academic and industrial labs. Its goal has been to characterize at least 15 GPCRs by 2015; it has already solved 13.

Interestingly, among the solved GPCR structures are those for the corticotropin-releasing hormone receptor and the glucagon receptor. Both have peptide ligands, and thus work by forming PPIs.

One company mentioned in the article, Heptares Therapeutics (Welwyn Garden City, UK), specializes in discovering new medicines that targeting previously undruggable or challenging GPCRs. In addition to discovering small-molecule drugs, Heptares, working with monoclonal antibody (MAb) leaders such as MorphoSys and MedImmune, is working to discover MAbs that act as modulators of GPCRs. Among Heptares’ targets are several GPCRs with peptide ligands.

Meanwhile, Kyowa Hakko Kirin Co., Ltd. has developed the MAb drug mogamulizumab (trade name Poteligeo), which is approved in Japan for treatment of relapsed or refractory adult T-cell leukemia/lymphoma. Mogamulizumab targets CC chemokine receptor 4 (CCR4).

Thus, aided in part by structural biology, the discovery of novel drugs that target GPCRs–including those with protein or peptide targets such as chemokine receptors–continues to make progress.

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.

In our  June 25, 2010 article on the Biopharmconsortium Blog, we discussed the “contrarian” views of Dr. Katherine M. Flegal and her colleagues at the National Center for Health Statistics of the Centers for Disease Control and Prevention (CDC) on the epidemiology of obesity.

According to Dr. Flegal, based on epidemiological data from the National Health and Nutrition Examination Survey (NHANES), people in the overweight class have a lower risk of death than those in either the normal weight or the obese class. These weight classes are determined on the basis of the body mass index (BMI), with underweight at <18.5, normal weight at 18.5-24.9,  overweight at 25-29.9, and obesity at >30.

Dr. Flegal’s conclusions–as summarized in our 2010 article–were mainly based on work published in the 2005-207 period, as well as other analyses of her results published between 2005 and 2010. In January 2013, Dr. Flegal and her colleagues published a report in the Journal of the American Medical Association. This report was based on an analysis of a wide variety of published reports indexed in PubMed and EMBASE that reported all-cause mortality for weight categories based on standard BMI categories.

In this study, the researchers compared all-cause mortality in the normal weight class (BMI 18.5-24.9) with that in the overweight (BMI 25-<30), grade 1 obese (BMI of 30-<35) and grade 2 and 3 obese (BMI of ≥35) classes. They came to similar conclusions as in their earlier studies. Specifically, both obesity (all grades) and grades 2 and 3 obesity were associated with significantly higher all-cause mortality as compare to normal weight. Grade 1 obesity was not associated with higher all-cause mortality, and overweight was associated with significantly lower all-cause mortality.

Reactions to Dr. Flegal’s 2013 study

As usually happens when one of Dr. Flegal’s “contrarian” studies is published, other leaders of the obesity epidemiology and nutrition community who hold the “majority” view react strongly against it. This was detailed, for example, in a feature article  in the 23 May issue of Nature written by science writer Virginia Hughes. On 20 February 2013, a meeting was held at the Harvard School of Public Health “to explain why [Dr. Flegal’s new study] was absolutely wrong”. The organizer of the meeting, Dr. Walter Willett, said in an earlier radio interview, “This study is really a pile of rubbish, and no one should waste their time reading it.” At the meeting, speaker after speaker got up to criticize the Flegal study.

The major concern of Dr. Willett and the other speakers was that Dr. Flegal’s study (and the commentary on that study in the popular press) would serve as a license for the general public and for doctors to let up on weight loss programs, and to undermine public policies aimed at curbing the rate of obesity. Dr. Willett was also concerned that the Flegal studies might be “hijacked by powerful special-interest groups, such as the soft-drink and food lobbies, to influence policy-makers”.

Nevertheless, as also detailed in Ms. Hughes’ article, other researchers accept Dr. Flegal’s results, and see them as part of the evidence for what they call “the obesity paradox”. Although for the general population overweight increases one’s risk of type 2 diabetes, cardiovascular disease, and cancer, overweight in some populations may not be harmful and may even lower the risk of death. These populations especially include people over 50 and especially those over 60 or 70, as well as patients with cardiovascular disease or cancer. We also discussed the decreased association of mortality with weight in older people in our June 25, 2010 article.

Explaining the “obesity paradox”, and the need for better metrics than BMI

In the 23 August issue of Science, Rexford S. Ahima, M.D., Ph.D. and Mitchell A. Lazar, MD., Ph.D. (both metabolic disease researchers at the Perelman School of Medicine, University of Pennsylvania, Philadelphia PA) published a Perspective entitled “The Health Risk of Obesity—Better Metrics Imperative”. The goal of this essay was to enable researchers to find better means to study and to explain the “obesity paradox”, and to use the results of their studies to improve the health of patents with metabolic diseases and their complications (e.g., cardiovascular disease).

These researchers noted that although it is easily measured and widely used, BMI does not adequately measure body composition (especially the proportion of muscle and fat) and the distribution of fat in the body. These factors may be especially important for such health outcomes as development of insulin resistance and type 2 diabetes, and cardiovascular risk. Other researchers, notably Dr. José Viña and his colleagues at the University of Valencia in Spain, who wrote a critical response to Dr. Flegal’s 2013 article, came to similar conclusions. The Spanish researchers criticized Flegal’s studies because they were based on BMI. However, unlike Dr. Willett, they accept the validity of the “obesity paradox”.

Notably, the Ahima and Lazar article includes a figure that shows metabolically healthy people with  normal and obese BMIs, and contrasts them with metabolically unhealthy people with normal and obese BMIs. The main difference between metabolically healthy versus unhealthy people (whatever their BMI) is muscle mass and fitness. The unhealthy subjects exhibit muscle loss, or sarcopenia, and reduced fitness.

The authors note that skeletal muscle accounts for the majority of glucose disposal. Thus loss of muscle mass, or sarcopenia, due to aging and/or physical inactivity, can result in reduced insulin sensitivity, development of diabetes, and poor cardiovascular health. This applies people with poor metabolic health, whether they have apparently normal BMIs or are obese. Metabolically unhealthy individuals–whether of normal BMI or obese–also have excess visceral fat. Excess visceral fat is associated with the metabolic syndrome and development of type 2 diabetes and cardiovascular disease.

Drs. Ahima and Lazar call for better metrics than BMI, in order to assess a patient’s risk of metabolic disease. They cite the “body shape index”, which quantifies abdominal adiposity (and thus visceral adiposity) relative to BMI and height as potentially a better predictor of mortality than BMI. The body shape index is based on measuring waist circumference, and adjusting it for height and weight. They further call for the development of “accurate, practical, and affordable tools to assess  body composition, adipose hormones, myokines, cytokines, and other biomarkers” to use in assessing obesity and other metabolic disorders in order to determine the risk of developing diabetes and cardiovascular disease, and the risk of mortality.

Appreciating the role of muscle mass in health and disease

The analysis of Ahimsa and Lazar also suggests the hypothesis that loss of muscle mass–sarcopenia–due to aging and/or lack of exercise may be a key factor in the development of obesity-related diseases.

There are at least two other recent reports that focus on sarcopenic obesity. The first, a 2012 paper in Nutrition Reviews entitled “Sarcopenic obesity in the elderly and strategies for weight management” is authored by Zhaoping Li, M.D., Ph.D. and David Heber, M.D., Ph.D. of the Center for Human Nutrition, David Geffen School of Medicine, University of California at Los Angeles. The second paper, entitled “Sarcopenic obesity: strategies for management”, by Melissa J. Benton, PhD, MSN and her colleagues (Valdosta State University College of Nursing, Valdosta, GA) was published in 2011 in the American Journal of Nursing. The first of these reports is a scientific review article, while the second is a practically-oriented report for nurses (carrying continuing education credits); the lead author is a nurse with advanced training in education, sports medicine, and gerontology.

The Li and Heber paper covers much of the same ground as the Ahimsa and Lazar Science Perspective, with respect to the inadequacy of BMI as a metric for obesity, and the need to have better measures of body composition (especially with respect to fat versus skeletal muscle). However, it goes beyond this concern for metrics, by focusing on “sarcopenic obesity”, its relationship with a sedentary lifestyle and with aging, and how sarcopenic obesity might be treated.

Loss of muscle mass as a function of aging in sedentary individuals results in age-associated decreases in resting metabolic rate and muscle strength, and is also a major factor in decreases in activity levels.  These factors result in the decreased energy requirement in aging individuals. If (as is usual) calorie intake does not decrease to match the decreased energy requirements, obesity (i.e., accumulation of excess body fat) results. Sarcopenic obesity in aging individuals is associated not only with type 2 diabetes and other metabolic and cardiovascular diseases, but also with loss of independence and increased risk of mortality. It is a major public health challenge in the over-65 population.

Li and Heber discuss various means to measure body composition, and thus to diagnose sarcopenia and sarcopenic obesity. They then go on to discuss ways to treat this condition, via emphasizing resistance training and increased intake of protein, in order to increase muscle mass and the resting metabolic rate. The authors cite resistance training as “the most effective intervention for reversing sarcopenia in the elderly”. Based on evidence in the field, the authors also hypothesize that increased dietary protein (especially the use of protein supplements or meal replacements) is also important in building muscle mass and as a result reducing fat mass.

It is known that increased dietary protein results in maintenance of muscle mass during calorie-restricted diets, as compared to diets with “normal” or inadequate intakes of protein. However, the authors see the need for more research to determine whether a high-protein diet (up to 35% of caloric intake) will be beneficial in improving muscle anabolic responses to resistance exercise in older adults.

The Benton et al. paper also emphasizes the role of resistance training and a high-protein diet in treatment of sarcopenic obesity. However, being a practically-oriented nursing article, it gives specific recommendations for exercise, as well as sources of high-quality protein in the diet. (This article focuses on high-protein foods, not protein supplements.)

This article also states that nurses should be knowledgeable about sarcopenic obesity and its management. They should also educate older patients on utilizing resistance training and dietary protein to prevent or reverse sarcopenia and sarcopenic obesity. This education should also apply to educating now-healthy aging adults on the need to prevent these conditions, since prevention is easier than reversing sarcopenic obesity once it has developed.

It would seem that not only nurses, but also primary care physicians and other doctors need to be aware of these issues as well.

The Benton et al. paper also wisely counsels that patients contemplating diet and exercise programs such as recommended in their article should first consult with their primary care physician. We agree with this recommendation. We also once again emphasize that this blog does not exist to give diet or exercise advice, or to receive comments or guest posts that purport to give such advice.

However, you are welcome to use this article, or better yet the publications we have cited herein, to help your primarily care provider to be aware of issues involving sarcopenic obesity. Some medical facilities also include physical therapists and/or access to gyms with trainers who can help patients with exercise programs, once one’s primary care physician has been consulted.

Conclusions

1. Currently marketed drugs for obesity–and for such conditions as type 2 diabetes, dyslipidemia, and other metabolic diseases that are usually found in obese individuals and metabolically unhealthy individuals with normal BMI–are generally prescribed as adjuncts to diet and exercise. “Diet and exercise” generally means the types of hypocaloric diets and aerobic exercise conventionally prescribed for weight loss. Researchers and physicians may need to take sarcopenic obesity into account when prescribing these drugs for patients with this condition, and in designing and conducting clinical trials. Diet and especially exercise recommendations may be different for patients with sarcopenic obesity than the current recommendations.

2. We have discussed “alternative” (i.e., non-CNS  or gut targeting) antiobesity therapies now in development in several articles on this blog. Unlike CNS-targeting drugs [e.g., lorcaserin (Arena’s Belviq) and phentermine/topiramate (Vivus’ Qsymia)], which are aimed at curbing appetite, these novel therapeutics are designed to increase energy expenditure or to inhibit the biosynthesis of fat. These drugs, if and when they are approved, will be indicated for patients with extreme obesity, such as those who may currently be candidates for bariatric surgery.

Similarly, we have discussed Novartis’ bimagrumab, an anti-muscle wasting drug now entering Phase 3 clinical trials in patients with the rare muscle wasting disease sporadic inclusion body myositis (sIBM). Bimagrumab is also in Phase 2 clinical trials in sarcopenic older adults with mobility limitations. If and when this drug is approved, it will be at least initially indicated for patients with sIBM, and perhaps eventually for older adults with severe sarcopenia (with or without obesity) that has resulted in mobility limitations.

It will be an extremely long time–if ever–before such drugs are approved for the broader obese and obese-sarcopenic population (or those at risk for these conditions). The diet and resistance exercise approaches discussed in this article may be appropriate for many in this broader group of individuals, and are free of drug-related adverse effects. They may also prevent the development of extreme obesity and its complications, as well as loss of independence due to sarcopenia or obese sarcopenia.

3. We have also discussed the development of anti-aging therapies in various articles in this blog. This field has generated a lot of interest in the news lately, because of Google’s launch of the anti-aging company Calico. As we discussed, for example, in our August 15, 2013 aging article, no pharmaceutical company can run a clinical trial with longevity as an endpoint. Companies must test their drugs against a particular aging-related disease. Many such companies test their agents (e.g., drugs that target sirtuins) against type 2 diabetes.

Why develop an “anti-aging” drug for type 2 diabetes rather than a specific antidiabetic drug? The hope is that an “anti-aging” drug approved for treatment of, for example, type 2 diabetes, will have pleiotropic effects on multiple diseases of aging, and will ultimately be found to increase lifespan or “healthspan” (the length of a person’s life in which he/she is generally healthy and not debilitated by chronic diseases).

Given the major role of sarcopenia and sarcopenic obesity in aging-related disability and mortality, those involved in research and development of anti-aging therapeutics need to take preservation and restoration of muscle mass into account, as they study and/or target pathways involved in aging and longevity.

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.

Skeletal muscle.

On August 20, 2013, Novartis announced in a press release that the FDA had granted breakthrough therapy designation to its experimental agent BYM338 (bimagrumab) for treatment of the rare muscle wasting disease sporadic inclusion body myositis (sIBM).

sIBM is a rare–but increasingly prevalent–disease. It is the most common cause of inflammatory myopathy in people over 50. sIBM has a yearly incidence of 2 to 5 per million adults with a peak at ages 50 to 70, with male predominance. Muscle wasting caused by sIBM is superimposed upon the sarcopenia (degenerative loss of muscle mass) that typically occurs with aging.

sIBM is characterized by a slowly progressive asymmetric atrophy and weakness of muscles. Typically, patients become wheelchair bound within 10 to 15 years of onset. Death may occur due to falls, respiratory infection, or malnutrition.

The causes of sIBM are not well-understood. In sIBM, an autoimmune process and a degenerative process appear to occur in muscle cells in parallel. In the autoimmune process, T cells that appear to be driven by specific antigens invade muscle fibers. In the degenerative process, holes appear in muscle cell vacuoles, and inclusion bodies containing abnormal proteins are deposited in muscle cells.

Despite the lack of understanding of the causes of sIBM, in recent years researchers have developed potential therapeutic approaches to this disease. These therapeutic strategies are based on the hypothesis that enhancing muscle regeneration can be beneficial in treating muscle-wasting diseases regardless of their cause. Researchers have thus been working on several approaches, principally 1. developing drugs that stimulate myofiber regeneration, and 2. cell-mediated therapies to replace damaged myofibers (e.g., autologous stem cell therapy). It is the first approach that led to the discovery of Novartis’ bimagrumab.

The myostatin pathway

Myostatin is a regulator of muscle growth in mammals and other vertebrates. It is a secreted protein that is a member of the transforming growth factor beta (TGF-β) family. It is secreted in an inactive form, and must be activated via cleavage by a metalloproteinase. The activated myostatin then binds to its receptor, activin receptor type IIB (ActRIIB). The binding of myostatin to ActRIIB on myoblasts initiates an intracellular signaling cascade, which (as with other members of the TGF-β family), includes activation of transcription factors of the SMAD family. The SMADs (SMAD2 and SMAD3) in the myostatin pathway go on to induce myostatin-specific gene regulation, which inhibits the proliferation of myoblasts and their differentiation into mature muscle fibers.

Bimagrumab, the myostatin pathway, and muscle-wasting diseases

Bimagrumab is a novel, fully human monoclonal antibody (MAb), which was developed by the Novartis Institutes for Biomedical Research (NIBR), in collaboration with the human MAb specialist company MorphoSys (Martinsried, Germany). MorphoSys’ HuCAL (Human Combinatorial Antibody Library) technology was used to identify bimagrumab.

Bimagrumab binds with high affinity to the ActRIIB receptor, thus blocking myostatin binding. The researchers working on development of bimagrumab hypothesized that treatment with the drug would have the same physiological result as myostatin deficiency. For example, myostatin knockout mice have a two-fold to three-fold increase in muscle mass, without other abnormalities. Humans with a loss-of-function mutation in myostatin exhibit marked increase in muscle mass, as well as increased strength. These findings suggest that a myostatin receptor antagonist such as bimagrumab would be a potent stimulator of muscle growth.

According to Novartis’ press release, this hypothesis has been borne out in human studies. The FDA granted breakthrough status for bimagrumab based on the results of a Phase 2 proof-of-concept (POC) study that showed that the drug substantially benefited patients with sIBM compared to placebo. The results of this study will be presented at the American Neurological Association meeting on October 14, 2013. Novartis also expects to published the results of the study in a major medical journal later in 2013.

In addition to sIBM, Novartis is developing bimagrumab for the common muscle-wasting disease of aging sarcopenia, as well as for cachexia in cancer and in chronic obstructive pulmonary disease (COPD) patients, and for muscle wasting in mechanically ventilated patients. In particular, the company is sponsoring a Phase 2 POC study (Clinical Trial Number NCT01601600) of bimagrumab in older adults with sarcopenia and mobility limitations. The study is designed to determine the effects of the drug on skeletal muscle volume, mass, and strength and patient function (gait speed). It will also generate data on the safety, tolerability, and pharmacokinetics of bimagrumab in older adults, as well as (via an extended study duration) the stability of drug-induced changes in skeletal muscle and patient function.

As we discussed in the Conclusions section of our August 15, 2013 blog article on aging, aging-related sarcopenia is a major causes of disability and death. We also said in that section that sarcopenia is not normally a target for drug development. At that time, we did not know about Novartis’ development of bimagrumab. We are happy to be proven wrong about drug development for sarcopenia.

Another approach to myostatin pathway-based drug development

A fully-human anti-myostatin MAb, Regeneron/Sanofi’s REGN1033 (SAR391786), is in Phase 1 clinical development for treatment of sarcopenia. Unlike bimagrumab, which binds to the myostatin receptor ActRIIB, REGN1033 binds directly to myostatin. REGN1033 thus represents an alternative approach to treatment of sarcopenia and other muscle-wasting conditions via the myostatin pathway.

Attempts to address the causes of muscle degeneration in sIBM directly

Despite the evidence from early clinical trials that therapies that enhance muscle regeneration may be effective in treating sIBM, some researchers believe that it will be necessary to identify the causes of muscle degeneration in sIBM and to address them. For example, there is evidence that in some patients, autoantibodies may recognize antigens that are enriched in regenerating muscle fibers. Some researchers therefore hypothesize that treating such patients with therapies that enhance muscle regeneration without addressing the autoimmune pathology may be counterproductive. Therefore, continuing research on the causes of muscle degeneration in sIBM and on potential therapies to slow this degeneration may still be important, despite the apparent progress of clinical trials of such drugs as bimagrumab.

For example, some researchers hypothesize that sIBM is a primary degenerative disease, like Alzheimer’s and Parkinson’s disease. As with these neurodegenerative diseases, some researchers have found evidence that misfolded proteins may be involved in the pathogenesis of sIBM. This avenue of research has led to the hypothesis that agents that enhance correct protein folding may slow muscle degeneration in sIBM patients. One such agent, CytRx’ arimoclomol has been in clinical trials in sIBM patients. [Arimoclomol is also in clinical trials in patients with amyotrophic lateral sclerosis (ALS)].  Arimoclomol appears to act as a coinducer of chaperone proteins such as heat shock protein 70 (Hsp70). Chaperone proteins promote the correct folding of intercellular proteins.

In a small POC Phase 2a clinical trial in Europe, arimoclomol showed early signs of efficacy, in addition to being well tolerated. There was a trend toward slower degeneration in physical function, muscle strength, and right-hand grip muscle strength in arimoclomol-treated patients as compared to placebo over an 8-month period.

Other researchers are attempting to address the inflammatory aspects of sIBM. For example, there are early clinical trials in progress of  the-anti-lymphocyte agent alemtuzumab (Genzyme’s Campath/Lemtrada) and the anti-tumor necrosis factor agent etanercept (Amgen/Pfizer’s Enbrel).

Meanwhile, additional basic research on the causation of sIBM continues. Some of these approaches may eventually lead to additional drug discovery strategies for this disease.

However, whether or not muscle-enhancing therapies such as bimagrumab might provide adequate treatment for at least some classes of sIBM patients (without addressing the autoimmune and/or degenerative aspects of the causation of the disease) will depend on the results of late-stage clinical trials now in the planning stage.

Conclusions

The development of bimagrumab represents an example of Novartis’ pathway-based rare disease strategy. We discussed this strategy in our July 20, 2009 Biopharmconsortium Blog article. Novartis researchers note that in many cases rare diseases are caused by disruptions of pathways that are also involved in other rare diseases and/or in more common diseases. The researchers therefore develop drugs that target these pathways, and obtain POC for these drugs by first testing them in small populations of patients with a specific rare disease. Drugs that have achieved POC in this rare disease may later be tested in other indications (especially including more common diseases) that involve the same pathway.

As we discussed in our July 20, 2009 article, the first drug that Novartis developed by using this strategy is the interleukin-1β inhibitory MAb drug Ilaris (canakinumab). The company conducted its first clinical trials in patients with cryopyrin-associated periodic syndromes, (CAPS), a group of rare inherited auto-inflammatory conditions that are characterized by overproduction of IL-1β. In 2009, the FDA and the European Medicines Agency approved Ilaris for treatment of CAPS. Since that time, Novartis has been conducing clinical trials of canakinumab in such conditions as systemic juvenile idiopathic arthritis (SJIA), gout, acute gouty arthritis, type 2 diabetes, and several others. Canakinumab had also been tested in rheumatoid arthritis, but these trials have been discontinued.

In the case of bimagrumab, Novartis researchers are targeting the myostatin pathway. The strategy is to first target the rare disease sIBM, and to obtain POC in human studies in that disease. Novartis claims (and the FDA concurs with them) that they have obtained POC in sIBM, and the company plans to present the results of its POC clinical trial later this year, both in a scientific meeting and in a publication. Novartis then plans to complete development of bimagrumab for sIBM, while also developing the drug for other muscle-wasting conditions, especially the more common aging-related condition sarcopenia, which is becoming a major public health problem.

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.

Hypothalamic nuclei on one side of the hypothalamus, in 3-D. Source: Was a bee. http://bit.ly/13o91HU

The Biopharmconsortium Blog has been following novel developments in anti-aging medicine and biology for several years. Much of the interest in this field has centered around sirtuins and potential drugs that modulate these protein deacetylase enzymes. Recently–on May 29, 2013–we published our latest blog article on sirtuins.

However, we have long been aware that studies in the biology of aging reveal that lifespan is controlled by sets of complex, interacting pathways. Sirtuins represent only one control point in these pathways, which might not be the most important one.

Now comes a research article in the 9 May 2013 issue of Nature, on the role of inflammatory pathways in the hypothalamus of the brain in the control of systemic aging. This article (Zhang et al.) was authored by Dongsheng Cai, M.D., Ph.D. of the Albert Einstein College of Medicine (Bronx, NY) and his colleagues. The same issue of Nature contains a News and Views mini-review of Zhang et al., authored by Dana Gabuzda, M.D. and Bruce A. Yankner, M.D., Ph.D. (Harvard Medical School).

The role of the neuroendocrine system–and other tissues–in the regulation of lifespan

The News and Views review begins with the statement that classic studies of aging-related pathways in Caenorhabditis elegans by such pioneering researchers as Cynthia Kenyon and Leonard Guarente, as well as later studies in Drosophila suggested that genetic changes that affect the function of nutrient-sensing and environmental-stress-sensing neurons can regulate aging of the entire organism.

In our May 11, 2010 Biopharmconsortium Blog article that reviewed the aging field, we focused on biochemical pathways that may affect lifespan, not the specific tissues in which they act. That article included a link to a 25 March 2010 review in Nature by Dr. Kenyon, entitled “The genetics of ageing”. As we said in our article, that review “discussed the panoply of aging-related pathways in worms, flies, and mice, especially the insulin/insulin-like growth factor-1 (IGF-1) and TOR pathways, as well as several other biomolecules and biological processes”. However, Dr. Kenyon’s article, and several of the references she cites, also deal with the tissues in which these pathways act.

Numerous leading researchers have found evidence that IGF signaling in the C. elegans nervous system regulates longevity. A 2008 article by Laurent Kappeler (INSERM U893, Hopital Saint-Antoine, Paris, France) and his colleagues referenced in the Kenyon review also found evidence that IGF-1 receptors in the brain control lifespan and growth in mice via a neuroendocrine mechanism.

Nevertheless, there is evidence that the insulin pathway in such tissues as the intestine of C. elegans can also regulate lifespan in a non-cell autonomous manner. These studies indicate that changes in insulin pathway gene expression in one tissue–perhaps with certain tissues (the neuroendocrine system, endoderm, adipose tissue) being especially important–results in coordinated changes in insulin pathway activity among all the relevant tissues of the organism. These studies complicate the determination that the neuroendocrine system is the locus of longevity regulation by the insulin pathway and other aging-related pathways.

The potential role of the hypothalamus in the regulation of mammalian aging

Based on the results of studies of neural regulation of lifespan in C. elegans and Drosophila, Zhang et al. asked whether the hypothalamus may have a fundamental role in the process of aging and in regulation of lifespan. The hypothalamus is a region of the brain that is critically involved in regulating such functions as growth, reproduction and metabolism. An important function of the hypothalamus is to link the nervous system to the endocrine system via the pituitary gland, an endocrine gland that is intimately associated with the hypothalamus, and is the master regulator of the endocrine system. According to the Gabuzda and Yankner News and Views article, the mammalian hypothalamus has similar functions to the nutrient-sensing and environmental-stress-sensing neurons of C. elegans and Drosophila that have been implicated in regulation of aging in those organisms.

Zhang et al. studied the increase in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in the hypothalamus of mice as a function of aging. NF-κB is a transcriptional regulator that is involved in cellular responses to stress, and in particular mediates inflammatory responses; it has also been implicated as a driver of aging-related gene expression in mice. The researchers found that the numbers of microglia (central nervous system cells that functionally resemble macrophages) in the hypothalamus increased as the mice aged. These microglia exhibited inflammatory function, expressing activated NF-κB and overproducing tumor necrosis factor alpha (TNFα). In turn, secretion of TNF-α (an inflammatory cell-signaling molecule) stimulated NF-κB-mediated signaling in hypothalamic neurons.

Zhang et al. showed–via using genetic models such as specific gene knockouts–that activation of the NF-κB pathway in hypothalamic neurons accelerated the aging process and shortened lifespan. Conversely, inhibiting the NF-κB pathway resulting in delayed aging and increased lifespan. They further showed that activation of neural NF-κB signaling resulted in declines in gonadotropin-releasing hormone (GnRH) levels. Since GnRH stimulates adult neurogenesis in the hypothalamus and hippocampus, decline in GnRH secretion suppressed neurogenesis. Conversely, hypothalamic administration of GnRH reversed aging-associated declines in neurogenesis.

The researchers also treated old mice with GnRH peripherally (i.e., via subcutaneous injection, rather than via hypothalamic administration). Such treatment with GnRH resulted in amelioration of aging-related changes in muscle, skin, and the brain. Notably, GnRH treatment resulted in amelioration of  aging-related cognitive decline. The researchers hypothesize that peripherally-administered GnRH (a neurohormone that is secreted by specific neurons in the hypothalamus) exerts its anti-aging effects via its action on one or more of the GnRH-responsive brain regions that lack a blood–brain barrier, such as the median eminence, subfornical organ and area postrema.

As pointed out by Gabuzda and Yankner in their News and Views article, a 2011 study showed that dendrites of hypothalamic GnRH-producing neurons extend through the blood–brain barrier. These dendrites are able to sense inflammatory and metabolic signals in the blood. Gabuzda and Yankner hypothesize that inflammatory signals in the periphery (which are known to be associated with aging, and such aging-related conditions as insulin resistance, obesity, and cardiovascular disease) may feed back via these dendrites to downregulate GnRH production in the hypothalamus. Such a feedback loop might be analogous to the coordination between peripheral and neural tissues of aging-related pathways seen in C. elegans by Dr. Kenyon and other researchers.

Despite these hypotheses (as pointed out by Zhang et al.), the mechanisms by which GnRH (especially peripherally-administered GnRH) exerts its anti-aging effects are not well-understood, and need further investigation. The researchers conclude, however, that the hypothalamus can integrate NF-κB-directed immunity and the GnRH-driven neuroendocrine system to program development of aging.

With respect to anti-aging therapy, the study of Zhang et al. suggests two potential therapeutic strategies–inhibition of inflammatory microglia in the hypothalamus, and restoration of levels of GnRH. Given the difficulties of specific targeting of microglia in the hypothalamus (across the blood-brain barrier), the second of these alternatives seems to be the more feasible of these strategies.

The GnRH receptor agonist leuprolide as a potential therapy for Alzheimer’s disease

GnRH has a short half-life in the human body, and thus cannot be used as a medication unless it is delivered via infusion pumps. However, the GnRH receptor agonist leuprolide acetate (AbbVie’s Lupron, Sanofi’s Eligard) has been approved by the FDA since 1985. It is available as a slow-release implant (AbbVie’s Lupron Depot) or a formulation delivered via subcutaneous/intramuscular injection. Leuprolide is approved for treatment of prostate cancer, endometriosis, fibroids, and several other conditions. Although leuprolide is the largest-selling GnRH agonist, there are other approved nanopeptide GnRH agonists, such as goserelin (AstraZeneca’s Zoladex) and histrelin (Endo’s Supprelin and Vantas).

As discussed in a 2007 review by Wilson et al., leuprolide acetate has also been under investigation as a therapeutic for Alzheimer’s disease (AD). Studies in mice indicated that leuprolide modulated such markers of AD as amyloid-β (Aβ) and tau phosphorylation, and prevented AD-related cognitive decline. For example, in a 2006 study in a classic mouse model of AD (Tg2576 amyloid precursor protein transgenic mice carrying the Swedish mutation) by Casadesus et al., the researchers demonstrated that leuprolide acetate halted Aβ deposition and improved cognitive performance.

Although Casadesus et al. attributed the efficacy of leuprolide to its suppression of the production of luteinizing hormone, Wilson et al. speculated that it might be possible that leuprolide works directly via GnRH receptors in the brain. Those receptors had only been identified in 2006 by the first author of the review, Andrea Wilson, and her colleagues at the University of Wisconsin. The direct action of leuprolide on GnRH receptors in the brain to ameliorate aging-related cognitive decline–and perhaps AD itself–is consistent with the 2013 findings of Zhang et al.

Development of a leuprolide-based Alzheimer’s disease treatment by Voyager Pharmaceuticals

As discussed in the review of Wilson et al., a Phase 2 clinical trial in women with mild-to-moderate AD receiving acetylcholinesterase inhibitors and implanted subcutaneously with leuprolide acetate showed a stabilization in cognitive decline at 48 weeks. A subsequent study in men (clinical trial number NCT00076440) was documented on ClinicalTrials.gov between 2004 and 2007. However, no results of this trial were ever posted.

The clinical development of a formulation of leuprolide (known as Memryte) as a treatment for AD had been carried our by a small Durham, NC biotech company called Voyager Pharmaceutical Corporation. Memryte was a biodegradable implant filled with leuprolide acetate, designed to treat mild to moderate AD.

After struggling to develop their treatment for nearly a decade, Voyager ran out of money, and stopped its R&D operations in 2007. In 2009, Voyager acquired a new set of investors, and changed its name to Curaxis. In 2010, Curaxis did a reverse stock merger, which enabled it to become a publicly traded company. Also in 2010, Curaxis attracted $25 million from a Connecticut investment firm. Nevertheless, Curaxis noted in its SEC filing that it would need “at least $48 million through 2014” to complete development of Memryte and file a New Drug Application (NDA) with the FDA.

However, Curaxis failed to find the needed funding and/or a partner to complete its development plans. In July 2012, the company filed for bankruptcy.

The failure of Voyager/Curaxis as a company does not necessarily mean that leuprolide may not be a viable treatment for AD. However, as we noted in earlier Biopharmconsortium Blog articles, development of an AD therapy is an enormously long, expensive, and risky proposition, which is beyond the capacity of a small biotech (such as Voyager/Curaxis) unless it attracts a Big Pharma partner. Moreover, treating AD once it reaches the “mild to moderate” stage is unlikely to work.

As we discussed in our April 5, 2013 article, the FDA has been working with industry and academia to develop guidelines for clinical trials of agents to treat the very earliest stages of AD, before the development of extensive irreversible brain damage. If another company endeavors to develop a formulation of leuprolide (or another GnRH pathway activator) for AD, it would probably do best to aim to treat very early-stage disease using the new proposed FDA guidelines.

Conclusions

Why should drug discovery and development researchers and executives be interested in anti-aging research? No pharmaceutical company will be able to run a clinical trial with longevity as an endpoint. However, the hope is that an “anti-aging” drug approved for treatment of one disease of aging will have pleiotropic effects on multiple diseases of aging, and will ultimately be found to increase lifespan or “healthspan” (the length of a person’s life in which he/she is generally healthy and not debilitated by chronic diseases). Numerous pharmaceutical and biotechnology companies have been working on discovery and development of treatments for major aging-related diseases, such as type 2 diabetes and AD. It would be truly spectacular if a new drug for (for example) type 2 diabetes would also be effective against AD.

Moreover, their are also major aging-related conditions, such as sarcopenia (aging-related loss of muscle mass, quality, and strength) that are not normally targets for drug development, but are major causes of disability and death. It would also be amazing if an anti-aging drug aimed at (for example) AD would be effective against sarcopenia as well. Note that Zhang et al. showed that GnRH treatment ameliorated aging-related changes in muscle in their mouse models. (Update: The statement that sarcopenia is “not normally a target for drug development” is no longer true. See our September 4, 2013 article on this blog, “Novartis’ Breakthrough Therapy For A Rare Muscle-Wasting Disease”.)

The study of Zhang et al. constitutes a new approach to anti-aging biology and target and drug discovery. However, aging biology is complex and not well-understood. Researchers will need to study many aspects of aging-related pathways for anyone to be able to discover and develop successful anti-aging drugs. This includes, of course, the sirtuin field, as well as the role of mitochondria in aging related metabolic imbalance, as exemplified by a recent paper in Cell.  As discussed in our other Biopharmconsortium Blog articles on anti-aging biology and medicine, there are many other avenues for investigation as well.

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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.