From March 13 to 16, we attended the 13th Annual Rachmiel Levine Diabetes and Obesity Symposium in bright and sunny Pasadena, CA. The conference attracted several hundred attendees, with 48% of the audience identifying as scientists, 36% as clinicians, 11% as educators, and 5% as industry professionals. Throughout the week, speakers mainly focused on type 1 diabetes cures and basic science; however, the talks still spanned several interesting topics, with valuable sessions on type 2 diabetes therapies, possible new treatment targets, and diabetes devices, as well as notable presentations on controversies in clinical trials (including commentary on bardoxolone and incretins’ possible association with pancreatitis). Below we present our full report from the conference as well as themes that connected the varying sessions. We have highlighted in yellow a panel discussion not included in full in its respective day-of report, and we have highlighted in blue our five favorite presentations.
The Symposium featured nuanced commentary on why attempts to cure type 1 diabetes to-date have largely failed. Dr. Richard Insel (JDRF, New York, NY) gave a very thought-provoking presentation on optimal trial design for type 1 diabetes cure research. He noted several challenges that have contributed to the limited number of type 1 therapies, including our incomplete understanding of the disease pathogenesis, a need for more validated targets and pathways, the heterogeneity of the type 1 population, and poorly predictive preclinical models. Similarly, Dr. David Harlan (University of Massachusetts School of Medicine, Worcester, MA) posited that type 1 diabetes’ genetic, immunologic, and physiologic heterogeneity along with our poor understanding of the disease’s pathophysiology has thwarted recent attempts to either prevent or cure the disease. Dr. Mario Ehlers (Immune Tolerance Network, Seattle, WA) reviewed type 1 trials, noting that while some results have been disappointing, the trials have provided valuable lessons and have elucidated which pathways may play a role in type 1 diabetes.
The conference included several presentations on possible type 1 diabetes cures in preclinical development. Dr. Chantal Mathieu (Katholieke Universiteit, Leuven, Belgium) discussed preclinical results using a novel approach to cure type 1 diabetes: low-dose anti-CD3 combined with lactococcus lactis bacteria genetically modified to secrete pro-insulin and IL-10. Results for this approach look encouraging in mice and Dr. Mathieu hopes to begin clinical development soon. Dr. Eugene Brandon (ViaCyte, San Diego, CA) gave an in-depth look at ViaCyte’s beta cell progenitor encapsulation device, VC-01, noting that the company plans to begin a phase 1/2a trial of the VC-01 in 2014. Dr. Mihaela Stefan (Mount Sinai School of Medicine, New York, NY) described how interferon-alpha (IFNa) is a critical cytokine in the development of type 1 diabetes and explained that it could be the missing link between viral infections and the development of type 1 diabetes. Dr. Defu Zeng (City of Hope, Duarte, CA) expressed enthusiasm about the potential of inducing mixed chimerism as a way to reverse type 1 diabetes autoimmunity.
Only a couple of weeks after ATTD 2013, the Symposium featured a session dedicated to discussing the Artificial Pancreas. The session included early preclinical data from TheraNova’s feasibility study of intraperitoneal glucose sensing. The lag time results look encouraging and the company expects to start a clinical study in the next couple of months incollaboration with Drs. Howard Zisser (Sansum Diabetes Research Institute, Santa Barbara, CA) and Eric Renard (University of Montpellier, France). Dr. Frank Doyle III (University of California Santa Barbara, CA) reviewed positive preliminary data (first shared at ATTD) on ambulatory closed-loop systems with faster insulin delivery (Roche’s DiaPort and MannKind’s Afrezza). Bringing the hospital perspective, Dr. Kazuhiro Hanazaki (Kochi Medical School, Kochi, Japan) discussed inpatient glucose control with the Nikkiso Company’s STG-55, an artificial pancreas device that is already available in Japan. In randomized prospective trials, surgery patients undergoing tight glucose control with the STG-55 had significantly fewer infections and significantly shorter stays in the hospital.
Though much of the conference focused on preclinical research, speakers also participated in a notable session on clinical trial controversies in diabetes care. Dr. David Kendall (Eli Lilly, Indianapolis, IN) kicked off the session by offering his perspective on the ACCORD trial, noting that it represents an outlier and requires further investigation. He pointed out the clinical implications of the trial, specifically identifying patients that may not benefit from intensive glycemic control. Renowned DCCT/EDIC statistician Dr. John Lachin (George Washington University, Washington, DC) offered valuable perspective on the trials’ results, including 1) that variations in A1c explain 90-99% of the differential risk of complications between the conventional and intensive groups and 2) hypoglycemia is a “failure of the technology to implement intensive insulin therapy.” Meanwhile Dr. Robert Vigersky (Walter Reed National Military Medical Center, Washington, DC) gave what we thought was a superb overview of glycemic variability. While no long-term evidence has been generated from a randomized, controlled trial, Dr. Vigersky brought plenty of evidence to the table that correlates glucose variability with adverse outcomes – in our view, this reinforces the need to use CGM in clinical trials to generate information on glycemic variability and to provide more nuanced data on the tested therapy/device.
Dr. Vigersky provided insights into the risk of pancreatitis with incretin therapies. Dr. Vigersky emphasized that the recent database analysis appearing in JAMA Internal Medicine should not impact the decision to prescribe the drugs. He described how such analyses are only reliable if HCPs accurately code their cases. The researchers used codes to control for a patient having obesity and alcoholism, which are both associated with increased risk of pancreatitis. Dr. Vigersky pointed out that many HCPs tend not to include codes for obesity or alcoholism, and instead focus on the codes for complications (which tend to be reimbursed more than obesity or alcoholism). Thus, these confounding variables were likely not well controlled for.
The most surprising presentation of the meeting for us came from Dr. Mark Cooper (Baker IDI Heart and Diabetes Institute, Melbourne, Australia), who shared details about Reata’s chronic kidney disease compound, bardoxolone. He believes that Reata “jumped into” the phase 3 BEACON study without having done the necessary basic science research. As a reminder, Reata and partner Abbott terminated the trial on October 18, 2012 due to safety concerns. Indeed, Dr. Cooper showed a preclinical study published just a few weeks later that revealed similar safety problems to those uncovered in the BEACON study. We were surprised to hear this and hope to learn more about the drug’s development path, what went wrong in BEACON, and if the company plans to pursue the compound further for diabetes. Further development appears unlikely at this point: aside from the press release announcing the trial’s termination, Reata and AbbVie have not commented on any of the above.
In addition to Dr. Cooper’s presentation, our four other favorites talks included Dr. Insel’s description of optimal type 1 diabetes clinical trial design. Dr. Harlan followed with a notable discussion of the pathology of type 1 diabetes and why past attempts to cure the disease have largely failed. The session on controversies in clinical trials featured two of our favorite presentations: Dr. Vigersky’s talk on the importance of glycemic variability and Dr. John Lachin’s talk on the nuances of DCCT/EDIC. We have highlighted the titles of our top five presentations in light blue.
- Executive Highlights
- Type 1 Diabetes Cure Therapies
- Clinical Trials for Type 1 Diabetes
- Debate: Is There Evidence for Distinct Courses in Loss of Beta-Cell Function in T1D?
- Plenary Lecture
- Diabetes and the Gut Microbiome
- Reversal of Autoimmunity and Transplantation Tolerance
- MESENCHYMAL STEM CELLS FOR THE TREATMENT OF DIABETES
- MULTIPOTENT MESENCHYMAL STROMA CELLS: POTENTIAL IN CLINICAL ISLET TRANSPLANTATION
- A NOVEL ORAL VACCINE FOR TYPE 1 DIABETES BASED ON LIVE ATTENUATED SALMONELLA
- REVERSAL OF AUTOIMMUNITY VIA INDUCTION OF MIXED CHIMERISM UNDER A RADIATION-FREE ANTI-CD3/CD8 CONDITIONING
- PANEL DISCUSSION
- Scientific Achievement Award Recipient
- Inflammation, Macrophages, and ER Stress
- Human Pancreas Development
- Diabetes Care and Drugs (Non-Cure Therapies)
- Clinical Trial Controversies in Diabetes Care
- Debate: The Beta Cell Will Be Preserved with Currently Available Therapy in Type 2 Diabetes
- Plenary Lecture
- Inflammation, Macrophages, and ER Stress
- Glucose Metabolism, Beta Cell Biology and Cancer
- Artificial Pancreas
- THE USE OF AN AUTOMATED GLUCOSE CONTROL SYSTEM FOR OVERNIGHT GLUCOSE CONTROL IN ADOLESCENTS WITH TYPE 1 DIABETES
- THE PERITONEAL CAVITY AS A SITE FOR GLUCOSE SENSING: A FEASIBILITY STUDY
- SYSTEM AND CONTROL ENGINEERING FOR TYPE 1 DIABETES
- INTENSIVE INSULIN THERAPY USING AN ARTIFICIAL PANCREAS WITH CLOSED-LOOP SYSTEM
- PANEL DISCUSSION
- Artificial Pancreas
- Diabetes and the Gut Microbiome
- Diabetic Complications
- Plenary Lecture
- Inflammation, Macrophages, and ER Stress
Type 1 Diabetes Cure Therapies
Clinical Trials for Type 1 Diabetes
OPTIMAL TRIAL DESIGN IN T1D
Richard Insel, MD (Juvenile Diabetes Research Foundation, New York, NY)
In his well-structured and engaging presentation, Dr. Richard Insel reviewed the challenges of conducting type 1 clinical trials and offered key recommendations. He began by noting that despite progress in the type 1 arena, no therapies have been shown to prevent type 1 diabetes or stop its progression. Several challenges have contributed to the limited number of type 1 therapies, including our incomplete understanding of the disease pathogenesis, a need for more validated targets and pathways, the heterogeneity of the type 1 population, poorly predictive preclinical models, the small commercial market for type 1 therapies, and the heavy resources and time needed for a type 1 clinical trial (details below). Dr. Insel suggested that in conducting future trials, researchers should perform initially exploratory clinical research (i.e., smaller and shorter trials), use intermediate endpoints, and consider host factors and population heterogeneity. Several themes emerged throughout his presentation including the need for biomarkers (and their potential value) and the benefit of following a comprehensive and cohesive clinical development strategy. Dr. Insel’s recommendations prompted us to think how such suggestions could also advance type 2 clinical trials and de-risk the drug development process. We believe that critically evaluating how trials are conducted and setting concrete goals will be key in driving progress on this front.
Dr. Insel opened with a brief overview of the current type 1 diabetes landscape. Beginning with the progress made, he noted that life expectancy has steadily increased, with diabetes patients recently diagnosed expected on average to live close to a normal lifespan. Trials such as the DCCT and its follow up have demonstrated that glycemic control can reduce the risk of type 1 diabetes complications. On the therapeutic front, cadaveric islet transplantation has been shown to induce insulin independence, with over 50% of patients recently being transplanted benefiting from five years of insulin independence. However, the procedure has several drawbacks including a shortage of available organs and the lifetime need for immunosuppression. New anti-VEGF therapies represent a dramatic advancement for diabetic retinopathy that not only arrest progression of vision impairment, but also can improve eyesight. However, despite this progress, several unmet medical needs remain: no therapies have been shown to prevent type 1 diabetes or to stop the progression of type 1 diabetes in the at-risk setting. While proof-of-concept studies show that drugs can prolong the honeymoon period transiently, no such drugs are clinically available. Furthermore, it remains uncertain how long beta cell function must be preserved in the new onset setting in order to provide clinically relevant benefits in the long term (e.g., better glucose control and decreased risk of complications).
Several obstacles have hindered the development of type 1 diabetes therapies. Scientists have a limited number of validated targets and pathways, as well as biomarkers for clinical trials. Furthermore, researchers often perform relatively isolated trials rather than following a comprehensive clinical development strategy. Dr. Insel noted that some type 1 therapies need to be applicable to not only adults but also to children, which raises the bar. He also pointed out that the type 1 diabetes market is itself relatively small and certainly less profitable than the type 2 market. However, he reasoned that the development of type 1 and type 2 therapies have significant overlap, and that the type 2 insulin-dependent population is larger than that for type 1. To Dr. Insel, limited commercial viability is “not a good excuse” for not pursuing type 1 therapies and the limited availability of new therapies.
Dr. Insel discussed the general challenges associated with conducting type 1 trials. He noted that researchers still do not fully understand the pathogenesis of human type 1 diabetes, remarking, “until we do, I think it will be a major obstacle to developing approaches to prevention.” The type 1 population is also quite heterogeneous, with some people progressing to overt diabetes and insulin dependence much faster than others. Dr. Insel emphasized the importance of determining whether a cycle of relapse and remission occurs in the at-risk setting, as this would influence therapeutic approaches. Type 1 trials are also challenging given their cost, size, and length (trials take at minimum one or two years), the lack of prognostic and predictive biomarkers, the difficulty in translating results from poorly predictive preclinical models, and the need for combination therapies for some indications.
Dr. Insel provided six recommendations for conducting type 1 trials.
1. Look at the big picture. Researchers should consider their long-term deliverables and how a single trial fits within an overall clinical development strategy. Researchers should also plan the next steps if the trial were to be successful.
2. The benefits of exploratory clinical research. Dr. Insel highlighted that prior to launching a phase 2 trial, researchers should consider conducting exploratory clinical trials – i.e., smaller and shorter trials that evaluate characteristics not rooted in efficacy. Such characteristics include a therapy’s mechanism of action, its dose and PK/PD profile, and the targets and pathways engaged at the molecular and functional level. Researchers can also use such trials to evaluate a pre-specified biomarker, or how different subsets of individuals respond to the therapy. Dr. Insel highlighted that companies are now frequently conducting exploratory trials to lower the risk of pursuing a type 1 therapy, and remarked that academic institutions should also adopt this strategy.
3. Using intermediate endpoints. Dr. Insel described the burden of conducting type 1 trials that last eight to ten years and explained that given our current knowledge of the disease progression, researchers should be able to conduct shorter trials that look at intermediate endpoints. Before turning to large size phase 2 trials, researchers can conduct shorter trials in the at-risk setting to answer key questions –e.g., whether the therapies can revert stable dysglycemia, preserve residual beta cell function, or even restore beta cell function. He qualified this statement by commenting that FDA approval will still likely require full-length trials evaluating whether the therapy will ultimately delay or prevent insulin dependence.
4. Applying information on type 1 diabetes pathogenesis. Researchers should consider the dialogue between beta cells and the innate and adaptive immune systems, as beta cells are active players in disease pathology. Dr. Insel also briefly discussed the honeymoon period, commenting that therapies need to be instituted sooner rather than waiting two or three months after diagnosis.
5. Taking into account host factors and heterogeneity. Dr. Insel highlighted the heterogeneity of the type 1 population and the need to consider patients’ age, duration of diabetes, genotype, and family type.
6. Leveraging biomarkers. Dr. Insel discussed the value of biomarkers: mechanistic biomarkers reflect whether a response has occurred and if the therapy hits the correct target, and are beneficial for dose-response studies; prognostic biomarkers predict which patients will progress in the absence of intervention and are helpful in evaluating benefit vs. risk; predictive biomarkers help elucidate which patients will respond to a therapy and are thus a key tool for personalized, precision medicine.
Questions and Answers
Q: May I challenge your message a little bit? Stimulated C-peptide is actually a very good biomarker in predicting beta cell mass in islet translation. So is there really a lack of good biomarkers or is there a lack of efficacy in the trials that we’ve been conducting?
A: I think it’s both. With respect to stimulated C-peptide, it obviously reflects functional beta cell mass, but it won’t tell us about anatomic mass. When we have stressed or dysfunctional or dedifferentiated beta cells, it gives us no insight into the total number of beta cells that are present. I would ideally love to have other types of biomarkers to look at those kinds of effects in a short period of time. I would posit that if we have a biomarker for stressed beta cells, we could see some kind of readout in a very quick period of time. I also want to point out that stimulated C-peptide is a kind of cumbersome assay. We could be doing something simpler.
Q: Animals models and human models speak in favor of C-peptide. If you have a patient on a normoglycemic level of control, then stimulated C-peptide is actually the best marker we have today of beta cell mass.
A There’s no question – it is the best marker. But even with your own work with regards to imaging, if we could image beta cells, I think it would be very useful in all the settings you noted as well.
Q: We are trying to measure effect, but we don’t see any positive outcomes. Should we blame the biomarkers, or the way we assay the outcomes of our trial? Or should we take a further step back? Perhaps the reason is that we are not targeting the disease mechanism.
A: We still don’t have a fundamental understanding of the pathogenesis of r type 1 diabetes and in its absence, we will struggle.
Q: You stated that we have no trials that have induced remission. So you don’t believe the Voltarelli trial?
A: I’m looking for approaches that are safe enough for use in children and adults, and that will be acceptable in the eyes of regulatory agencies. I don’t think that trial as originally performed will be accepted and adopted.
Q: I thought it has been accepted by the FDA?
A: To the best of my knowledge it has not started yet in Chicago.
Q: I still think that could be considered.
A: Would you consider it for your child?
Q: If it works, yes.
A: Is it safe? If a procedure has a mortality rate of one in 20o or one in 300 then…
Q: That’s not true. I do agree that safety is important, but there’s still a group that could benefit.
A: The trial tells us in a proof-of-concept manner that we can induce insulin independence in that setting. I would posit that we need to take the principles from that trial and learn how to apply them safely.
Q [different person]: It’s a recipe for failure – we have different mechanisms acting and if your compound is active in only one of these mechanisms, you may have a negative outcome. [Editor’s note: we were unable to catch this sentence]. If you can predict who will develop the complication, you can get a better risk/benefit analysis.
A: The future lies in precision medicine. We are going to have to tailor the right treatment to the right individual at the right time. We will need better predictive capabilities and we require new biomarkers for such. s. Although we have C-peptide, we don’t have good biomarkers to predict who is going to progress with respect to diabetic kidney disease. You’re right – biomarkers could d change the risk/benefit profile and will change how we practice medicine.
Q: Regarding the risk/benefit analysis – I don’t know if anyone has ordered insulin pills for their mice. There are also insulin pills for humans, but they are not approved in the US or Europe. But in some places in the world that’s the alternative therapy. Furthermore, diabetes management depends on socioeconomic interventions. What’s the acceptable risk for those with high socioeconomic status? They have approximately normal life expectancy. But we also have the 20 year olds with type 1 diabetes after a kidney transplantation.
A: I agree, the benefit/risk analysis has to be done with respect to the individual, the geography, the alternatives, etc. And I think that regarding the benefit/risk profile, it’s up to patient advocacy organizations such as JDRF to make the case and to advocate with respect to benefits and risks. But we can only do that based on data. It has to be a data-based discussion with regulatory agencies and I look to this community to generate that kind of data.
ALL RECENT-ONSET TRIALS LOOK THE SAME, OR DO THEY?
David Harlan, MD (University of Massachusetts School of Medicine, Worcester, MA)
Dr. David Harlan began his presentation explaining that he was going to be “like a politician” and not answer the question he was given. Instead he proceeded to give an engaging and nuanced description of what we do and – even more so – what we do not know about the pathophysiology and development of type 1 diabetes. He began by noting that we do not even know how many beta cells are within a person’s pancreas before they develop type 1 diabetes and that vast heterogeneity exists in pancreatic volume. Thus, he posed the question of whether people with type 1 diabetes may have fewer beta cells to begin with. He noted that contrary to historical belief, the beta cell is not a passive actor while an autoimmune reaction destroys it; instead, it plays an important role in its destruction through beta cell stress and that beta cells vary in how well they manage this stress. In terms of the potential for beta cells to regenerate, Dr. Harlan concluded that a person stops making more beta cells once they are about 25 years old. Furthermore, he found that nine months of exendin treatment in a study enrolling people with type 1 diabetes did not cause the pancreas to produce additional insulin. On a more positive note, he highlighted that the prognosis for type 1 diabetes has dramatically improved; the excess mortality associated with type 1 diabetes is ~2% over 20 years. Thus, he argued that new therapies must be safe in order to have a positive benefit:risk profile. However, he noted that the few preventative treatments for type 1 diabetes that have provided temporary benefit in clinical trials, had limited efficacy and intolerable safety profiles. He suggested that the genetic, immunologic, and physiologic heterogeneity associated with type 1 diabetes has thwarted these recent attempts to prevent or cure the disease. He concluded that we need a better understanding of the pathophysiology of type 1 diabetes if we are going to develop a cure for the disease.
Dr. Harlan stated that most recent trials of possible type 1 diabetes cures had negative results and that those with positive results were only transient and were often associated with poor safety profiles. Dr. Harlan concluded that the only difference he could find between the therapies that worked and those that did not was how aggressive of a cytokine storm they induced.
The therapies he listed as having recent negative results included: 1) oral insulin, 2) mycophynolate mofetil plus daclizumab, 3) GAD65-alum, 4) intranasal insulin,5) IL-2 plus rapamycin, and 6) canakinumab.
The five therapies he listed as transiently preserving beta cell function included: 1) teplizumab, 2) oxelizumab, 3) rituximab, 4) abatacept, and 5) autologous bone marrow rescue.
Dr. Harlan recommended that researchers studying human beta cell development, regeneration, and function use mice models of type 1 diabetes that have no immune system. Researchers can then introduce human stems cells with beta cell potential and human immune cells in order to more accurately replicate human type 1 diabetes.
Dr. Harlan posed the question of whether people with type 1 diabetes may have fewer beta cells to begin with. He noted that we do not even know how many beta cells are within a person’s pancreas before they develop type 1 diabetes, since we cannot easily or accurately measure islet mass in vivo or ex vivo. Additionally, vast heterogeneity exists in the number of beta cells people have. In one study there was a 2.5- to 3-fold variation in the pancreatic volume among people without type 1 diabetes.
He noted that children ages 12.4-14.7 years with type 1 diabetes tend to have a lower baseline C-peptide level than older age groups. He posited that this might be because they have fewer beta cells in their pancreas than people who develop type 1 diabetes later in life.
In a study of seven people diagnosed with type 1 diabetes 12-50 years earlier, Dr. Harlan found that their native pancreases all produced insulin but only in very small quantities. Notably, these patients had received a pancreas transplant 2-10 years earlier, meaning that they had been on immunosuppression for up to 10 years. Despite this very little insulin was produced, suggesting that either the patients’ beta cells did not regenerate or the beta cell stress was continuing to destroy beta cells.
Dr. Harlan presented a creative study that found that people stop producing beta cells around the age of 25 years old. The study takes advantage of the rapid increase in the amount of 14C in the atmosphere during the 1950s and 60s due to atmospheric nuclear testing. People’s cells incorporate carbon from the atmosphere – including 14C – into their DNA when producing a new cell. Thus, by looking at the content of 14C in a cell’s DNA one can date when that cell was created. Using this technique, the researchers were able to determine that people tend to stop making new beta cells around that age of 25 years old.
Dr. Harlan showed an analysis of the DPT1 that demonstrates that type 1 diabetes has a long prodrome. He remarked that people often believe that the DPT1 found no measurable decline in C-peptide levels up to the time of diagnosis. However, this new analysis found that some participants were progressors and some were non-progressors. Among the progressors C-peptide levels declined for at least the two years prior to diagnosis.
Pancreases are also very heterogeneous in their composition. Studies have found that donors who are younger than 40 years old tend to have pancreatic islets comprised of more beta cells than those who are older than 40.
Dr. Harlan noted that while we do know that type 1 diabetes has a genetic component much of genetics’ role is still not understood. He remarked that while we know that the MHC Class II is strongly associated with the development of diabetes, only one- third of people with type 1 diabetes have this risky allele.
He continued to remind the audience that we do not know what the triggering event is for a person developing autoimmunity. Dr. Harlan expressed some hope that that TRIGR and TEDDY will provide some answers (TRIGR is assessing the role of cow’s milk-based formula compared to hydrolyzed infant formula and TEDDY is assessing multiple infectious agents, dietary agents, and other environmental agents). We note that TEDDY’s estimated study completion date is September 2025 and TRIGR’s is February 2017 (ClinicalTrials.gov Identifiers: NCT00279318 and NCT00179777, respectively).
ANTI-CYTOKINE THERAPIES OVERALL
Mario Ehlers, MD, PhD (Immune Tolerance Network, Seattle, WA)
Dr. Mario Ehlers reviewed recent type 1 therapy trials with the overarching message that even failed trials provide valuable learnings. Dr. Ehlers discussed TrialNet’s Anti-CD20 and CTLA-4 Ig trials in new onset diabetes, in which both therapies preserved C-peptide for only roughly six months. Despite the disappointing results, each trial indicated that these biologic pathways may play a role in type 1 diabetes. He also noted that both TrialNet’s GAD-alum trial and Anti-IL1β trial in new onset diabetes showed no benefit with the drugs (results for the Anti-IL1β trial will be published in the Lancet this year). Dr. Ehlers then discussed several trials conducted by the Immune Tolerance Network (ITN), including one of IL-2/Rapamycin, which indicated that the combination therapy may produce transient beta cell dysfunction. Another study of Thymoglobulin showed no benefit with the drug (results will be presented at ADA 2013). Dr. Ehlers commented that despite its limitations, anti-CD3 antibody therapy was one of the most promising approaches and the ITN’s AbATE trial showed that responsiveness to the therapy was correlated with glycemic control and insulin use. In concluding his presentation, Dr. Ehlers highlighted that future trials should investigate combination therapies, use biomarkers to monitor patients’ progress, and consider more efficient trial designs.
- Dr. Ehlers noted that for the past ten to 15 years, type 1 trials have followed the standard model originally pioneered by TrialNet: a two-arm trial design that randomizes participants 2:1 to drug vs. placebo with an endpoint of stimulated C-peptide levels at either one or two years. The trials typically recruit 60-90 patients aged eight to 35 years with recent-onset type 1 diabetes and residual C-peptide function
Mario Ehlers, MD, PhD (Immune Tolerance Network, Seattle, WA); David Harlan, MD (University of Massachusetts School of Medicine, Worcester, MA); Richard Insel, MD (Juvenile Diabetes Research Foundation, New York, NY); Matthias von Herrath, MD (La Jolla Institute for Allergy and Immunology, La Jolla, CA)
Comment: I have a comment based on the European experience with anti-CD3. We did manage to recruit within six weeks of diagnosis. Even within these six weeks we found that people who began sooner did better. It is not a matter of months it is probably a matter of days.
Comment: On the dose of anti-CD3, if you titrate in NOD mice, there is a bell-shaped curve. You get a bigger effect as you increase the dose, however, at a certain point you lose that effect. It is correlated with depletion of your T regulator cells.
Comment: I was surprised that you were surprised IL-1 did not work. It did not work in the NOD mice, so I would have put my money on IL-1 not working.
Q: I also believe that antigen based therapies is the way to go probably in combination with immunomodulation. I also have the impression that it is a dangerous intervention. A question to the three of you, how will we always know that using an antigen-based therapy will not make the disease worse?
Dr. Insel: With respect to individuals who have type 1 diabetes, I’m skeptical that you can make it worse from the immune standpoint. I would say that if you’re trying to look at immune effect, look at them. Ignore the beta cell. Think like an immunologist. Go into the established type 2 setting. We have a couple caveats – it’s possible that an individual with a particular genotypes associated with type 1 diabetes may have a higher threshold. There may be more stringent requirements to induce immune tolerance in an autoimmune background. Even if something works in a non-autoimmune genetic background, it may not work in other backgrounds. My advice is to show that on a mechanistic level, you’re doing something. With respect to anti-immunoglobulin specific therapies, that’s how the body does it all the time. This will prove safer than immune modulation
Dr. Ehlers: From my point of view, one issue apart from going first into recently established disease or high-risk individuals is to take a careful look within antigen-accelerated disease testing. I think that the evidence that whole proteins have done this is relatively low. The other thing that has to be done in the antigen therapy field is that there ought to be data as to which antigens give the optimal response. That’s not trivial. You not only need evidence that you’ve done something with the antigen, but that the antigen has done something in the individual that you can correlate with a beneficial outcome in terms of diabetes. We will be able to do the strategies, but we have to be unified in this vision. These exercises are not trivial – they require a lot of resources and time. We should not have all the trials be fragmented.
Dr. Insel: On this idea that earlier is better, it is conceivable that there is a permissible window that then closes down. It may be that some beta cells do differentiate. Maybe there is a window in which they could redifferentiate if rescued but that if you do not hit that window they become alpha cells or die off. It also may be that at a certain point they cannot relieve the beta cell stress even if the autoimmunity is removed.
Q: The take-home message seems to be that even when we have disappointing outcomes, we can still learn a lot from the mechanistic standpoint. But I would ask how would we scientifically approach that? We heard from the lectures here that people participating in studies can be divided into responders and non-responders, but taking into account the heterogeneity of the disease, you should also divide the placebo group into responders and non-responders. And of course, the placebo group cannot respond to anything, but you will have in that group individuals with preserved C-peptide over the same period of time as the responder group. Shouldn’t we also do the same comparison to that subgroup in the placebo group to really have a scientifically sound comparison and learn from the experiments that you’ve conducted?
Dr. Ehlers: Yes that is very problematic. In AbATE, in a sense, we were fortunate. The placebo response was quite tight and 45% of those who were treated lost less than that. In other studies if you apply that same criteria, there is too much overlap in the placebo and treated group. I think that has to do with heterogeneity of age, race, gender, duration of diabetes etc. I agree that one of the key problems is that these so called responders inside the placebo group confound the issue; people who are called elite responders and would do ok regardless of whether they received treatment. It is difficult because you do not know if you have included some of those people.
Dr. Harlan: We know that there is tremendous genetic and beta cell mass heterogeneity. What if all the potential targets we can hit are right, but they are all right in only 5% of the patients, so that we never see the overall effect?
Dr. von Herrath: If we say that we want to carry out short, small, proof-of-concept trials in well characterized people. We need to think about if that is going to be feasible; we need to think about balance, because otherwise we will have an n of 500,000 as we try to capture the right subsets. Where are we going to hit the sweet spot, in terms of how many patients we would recruit for a trial like this, and what stratification strategy should we employ?
Comment: We’re in the modern generation of all these trials, which generally started around 2000. We made a decision to say, “We don’t know enough about heterogeneity in a clinical course” so we deliberately said “We want the same study criteria for participants.” We now have 10 years of trial data from multiple trials, which will now allow us to go back and see if we can find subsets that we can concentrate on. All that being said, anything you do in a small pilot study – as long as you don’t say that the drug is a miracle cure – is an important step in the process. It seems like one of the themes is that this is a whole hodgepodge of things with no overarching strategy. But I don’t think that is necessarily true. Those three therapies that have been shown to work at least to some extent in new onset diabetes are now being investigated for prevention. We now have a prevention trial testing three drugs. I’d make the pitch that for full-on prevention trials, we need to screen relatives of people with type 1 diabetes. We know that most families don’t know that family members have a 15-fold increased risk. We do new onset trials to get signal and safety information, and the ultimate goal is for prevention. We need everyone’s help in this audience to screen people for them.
Q: What is the presumed mechanism by which autologous bone marrow transplantation thwarts islet autoimmunity?
Dr. Harlan: It is supposed to reset the immune system. That is what they say. I would say that the scientific rationale for that study is no stronger or weaker than the others we have discussed. I think that the main concern is the safety associated with it.
Dr. Insel: We have to look at if there is a role for components of that regimen. Genzyme and others should think about trying to take this apart and really understand it.
Dr. Ehlers: A group is looking at combining two of the components of the Voltarelli cocktail. They show relative preservation of Treg, unlike what we saw in ATG monotherapy. That could explain why that combination is better than the monotherapy. It is a very toxic against effector cells, both B and T cell – it really takes them out. One thing that we could not do was make a dent in the T effector cells. It is a great drug but it has an unacceptable side-effect profile.
Q: Considering that people under age 25 have the greatest beta cell proliferative capacity, why isn’t that group targeted in most clinical trials?
Dr. Ehlers: We constantly have to justify why we do these trials in children. That’s the group where we see the most potential, where the great unmet medical need is. But we always face the ethical problem of at what point is it acceptable to evaluate new agents in people under 18? What is the risk/benefit ratio?
Dr. Insel: I would turn the question around and ask why would that group have the fastest fall-off in C peptide levels after insulin in the new onset setting.
Comment: I am not an advocate for this trial, but it does show that some combinations could work. Lets not forget that it is a proof-of-concept trial, showing that you can cause lasting remissions.
Dr. Insel: Of the original 22 participants, how many are still insulin independent?
Comment: It is hard to determine because some continued to receive transplants but the longest period of insulin independence has been seven years.
Dr. Insel: Most are back on insulin.
Comment: I do not want to advocate for it, however, those who are on insulin are on less.
Debate: Is There Evidence for Distinct Courses in Loss of Beta-Cell Function in T1D?
Carla Greenbaum, MD (Benaroya Research Institute at Virginia Mason, Seattle, WA)
Dr. Carla Greenbaum began the debate confidently arguing that there are indeed distinct courses in loss of beta cell function in type 1 diabetes. She grounded her arguments in three areas: differences in clinical presentation/outcomes, differences in beta cell function, and differences in pathophysiology (“speculative” and “a little wobbly”). Dr. Greenbaum emphasized that the answer to the debate’s question is very important for clinical trial design, something that is commonly taken into account in other autoimmune diseases like MS. In her view, we can increase the benefits of a therapy if we more narrowly define the population. Based on the voting at the end of the debate, Dr. Greenbaum won quite handily, persuading an additional 13% of the audience to vote “Yes.”
Patients with type 1 diabetes exhibit differences in clinical presentation and outcomes. Dr. Greenbaum showed a slide with many pictures of patients, noting that there is a whole range of different people that get type 1 diabetes. She also highlighted differences in clinical outcomes, which are underappreciated in her view – for instance, 3% of type 1 patients died within 20 years of diagnosis, 9% of patients on intensive therapy get cardiovascular disease within 30 years, 21% of patients on intensive therapy get proliferative retinopathy within 30 years, and 25% have hypo unawareness. In her view, these percentages suggest type 1 diabetes is different in different individuals.
- Longitudinal and cross-sectional data suggest there are differences in the decline in beta cell function among people with type 1 diabetes. She highlighted data published in Diabetes (Greenbaum et al., 2012) on 200 TrialNet placebo patients that documented reductions in C-peptide over two years. Dr. Greenbaum emphasized that the “pretty picture” (a linearly sloping decline) is a result of using a population mean. In looking at the raw data within each age group, she showed how patients differed in their declines in C-peptide. Overall, 93% of patients had some detectable C-peptide at two years and 66% of individuals exceeded the ≥0.2 pmol/ml C- peptide threshold established in the DCCT. In 11% of patients, there was no significant fall in C- peptide from baseline at the two-year mark. Differences in C-peptide were also found in the 2009 SEARCH study (Greenbaum et al., Diabetes Care) – within the first year of diagnosis, 83% of youth had a fasting C-peptide ≥0.23 ng/ml and 31% had values ≥1.0 ng/ml. Similar data has been observed in the T1D Exchange (submitted to ADA 2013). On the anatomy side, Dr. George Eisenbarth emphasized key differences in the type 1 diabetes pancreas in his Banting Lecture from a few years ago. Meanwhile, Dr. Peter Butler also recently published data (Diabetes 2013) documenting the wide range in the number of beta cells in deceased people without diabetes.
Regarding differences in pathophysiology, Dr. Greenbaum admitted that the evidence “is a little wobbly,” but data from other autoimmune disease is supportive. Borrowing from multiple sclerosis, she showed how the disease has distinct relapsing and remitting phases with subsequent progression. There are waves of peripheral activation and inflammation that are distinct from organ damage. Crohn’s disease is similar. She wondered whether this could this be the case in type 1 diabetes – for example, patients’ rate of fall in insulin secretion in the first year is different from the second year.
- The answer to the debate’s central question has important implications for trial design. Dr. Greenbaum again harkened to multiple sclerosis, where patients are typically classified into one of four different disease groups and offered different treatments. She postulated that this could be done in type 1 diabetes, even with something like immunologic markers (e.g., IL-2R signaling). Dr. Greenbaum emphasized the importance of entry criteria when designing a trial, especially for people at the extreme. In her view, we can increase the benefits of a therapy if we more narrowly define the population. We agree and hope to see more individualized trial designs targeted at very specific populations.
Kevan Herold, MD (Yale University, New Haven, CT)
Dr. Kevan Herold countered Dr. Carla Greenbaum by arguing that the variations in beta cell loss she described were not dramatic and as being due to chance encounters with triggers in the environment. He characterized the beta cell death experienced during prediabetes as occurring at a slow and steady rate with stochastic starts and stops. He detailed how these stochastic starts result from a pool of precursor cells being activated by inflammatory and other unknown triggers to induce beta cell killing (he mentioned viruses as one possibility). Thus, according to Dr. Herold, much of the variability in beta cell loss seen between people with type 1 diabetes can be explained by the timing and nature of these chance triggers. However, Dr. Herold argued that there is dramatic heterogeneity in people’s response to type 1 diabetes treatments. He detailed how multiple type 1 diabetes treatments are known to have responders and non-responders and that retrospective analyses have been able to determine characteristics, such as age and baseline A1c, which predispose a person to respond to certain treatments. Dr. Herold concluded that researchers must target treatments to people likely to respond, because in spite of there being little distinction in how type 1 diabetes progresses, there is dramatic variability in treatment responses.
Dr. Herold characterized the variations in beta cell loss seen as being not dramatic. For example, in an analysis of people’s insulin secretory responses during the first two years of having type 1 diabetes the standard deviation was only ~17%.
Considering the variability in the age of diagnosis, Dr. Herold argued that the reason some people develop type 1 diabetes earlier in life is because they have lower C-peptide levels at baseline. Thus, even though young and old people with type 1 diabetes progress similarly, it takes less time for them to have undetectable C-peptide levels.
Dr. Herold argued that there is variability in people’s response to possible type 1 diabetes cure therapies and that the more work needs to be done on matching people with the right therapy. For example, people in AbATE who responded to teplizumab (defined as losing less than 40% of their beta cell function) were more likely to have a lower A1c level and use less insulin at baseline than non-responders (i.e., people whose beta cell function was reduced by at least 40%). Responders had C-peptide levels that were an average three-fold higher than non-responders.
Carla Greenbaum, MD (Benaroya Research Institute at Virginia Mason, Seattle, WA); Kevan Herold, MD (Yale University, New Haven, CT)
Q: Would you like to take a moment to rebut your opponent?
Dr. Carla Greenbaum: My opponent puts lots and lots of data up there, hoping that you were sleeping and not following. He would point out that there were differences and then highlight the amount of overlap saying, “wasn’t there so much overlap?” So I think my opponent made my case for me because he did note the differences.
Dr. Herold: My opponent showed you the means for the various age groups. I guess what I would like to do is take up answer D: it doesn’t matter. The ship is going down. Whether the fall is 732 or 725 glycomols per ml per month, it doesn’t matter. It’s all going down. My argument, in some ways, is in agreement with my opponent. There are differences. But my point is that the baseline is the same. There are environmental exposures that move things along, but the background is the same.
Dr. Greenbaum: At the end of the day, the answer is not [I do not care]; you have to care. You are not going to submit people to therapies that are not benign, which they likely will not respond to. I note though that when we were assigned this topic to debate we had to discuss, which sides we were each going to take. We did not know which side we should each take. We should not leave anyone with the impression that this is something more than an artificial discussion, though an important one, going on here.
Dr. Herold: We had to confirm it, but we could have easily switched the sides around.
Q: Can you talk about the NPOD data on anatomy in type 1 diabetes? How do you reconcile that you're your positions? And what about GWAS data?
Dr. Herold: One of my slides has all the genetics data. Yes, you’re right. The genetic data would suggest that there are a number of different genes that seem to be contributing to the development of diabetes. Not one individual has all the genes, and different individuals have different numbers of genes. As we go deeper into the GWAS data, what I think it is going to show is that there are common pathways that are targeted. There maybe different genes, but if we go deeper and look at the networks, we might find that there are a number of similarities between individuals. On the NPOD data, this is always a hard one. I would just say that those people are dead. [Laughter]
Dr. Greenbaum: What my opponent is saying is that as fantastic a resource as that is, they are retrospective. Trying to figure it out is unclear – there are relatively small numbers available, so it’s hard to make any generalizations.
Dr. Roep: There was one generalization –not a single graft looked like the other. That is something that has become clear.
Dr. Herold: That doesn’t refute my argument. The islets may vary. There can be a process that differs from time to time – individuals are more than just one islet. They are lots of islets. I think you’re missing the forest from the trees.
Q: Why should the pancreas be the only organ in the human body affected by only one disease?
Dr. Herold: I would take it back to the model that I am suggesting. First of all there are autoimmune features in type 1 diabetes I do not think that it is fair to say that in all individuals it only effects one organ. I would go back to the model I am suggesting that the islets may be targeted by factors – maybe viruses maybe other unknowns that trigger the expression of antigen and recognition of antigens by the immune system. I do think that there is specificity in terms of targeting islets
Comment: People with kidney disease, liver disease, skin disease – you could not treat all of them with the same kind of the treatment. I think that we have to acknowledge that the pancreas is like every other organ in the body that is impacted by multiple diseases. We need to target people with the intervention for what disease they have. We are putting all of our patients in one trial so of course we do not see anything. I think it is vitally important that we will stratify people in our trials.
Dr. Herold: I agree with you completely about the immune therapy but it sounds like you are going to vote [yes].
Q: I’m wondering about diabetes development in people with one antibody. There must be some variation – why do some people continue to clinical diabetes, but most don’t develop the disease.
Dr. Herold: The easy answer is we don’t know the antigens that are being targeted. Antibodies don’t kill beta cells. T cells do. We’re not picking up other specificities.
Dr. Greenbaum: Single antibody positive people have less than a 3% risk of getting type 1 diabetes. Some people get LADA, maybe when they are 60 years old. But anybody who has two antibodies repeatedly will get diabetes. We’re almost 100% certain.
Q: I think you both are being imprecise with terms and I would argue that we are mixing function and mass repeatedly. This is a discussion that has been had for many years in type 2, so I would like for both of you to comment on whether you think that function and mass are in parallel. If you think there could be instances when mass could be maintained but function declines of the opposite?
Dr. Greenbaum: The easy answer is that nobody knows the answer. I think that there are going to be times when mass is maintained but function declines. At three-months after diagnosis, people have an increase in C-peptide and they certainly did not go grow more beta cells.
Comment: Well you think they did not grow more beta cells.
Dr. Greenbaum: True, I do not know. I think that they did not grow more.
Dr. Herold: I would agree with that 100%. We have mouse data where we can look at insulin staining and mass. I would say that what my opponent just said is exactly right. There is recovery of function without an increase in mass in the period after either immune therapy or diagnosis.
Dr. Greenbaum: The best data we have was baboon data, which found that function was best correlated with mass. That was in a non-immune model and had several limitations. So I don’t think we have another way to know.
Comment: I think that we can agree with him that there might be a disconnect there, which could play a role.
Dr. Greenbaum: But even if there is a disconnect and you do not regain function does it really matter?
Comment: I would argue that the trees and forest are bigger than type 1 diabetes. I study cystic fibrosis related diabetes. There are similarities regarding diabetic retinopathy, neuropathy, and nephropathy. The differences are in the immunologic process. Cystic fibrosis related diabetes is due to sub-acute chronic lung disease. Cytokine levels are five fold greater than what you see in type 2 diabetes. if you look closely at all three diseases, there are different immunological responses. Obviously, there are similarities in glucose toxicity.
Is there evidence of distinct courses of beta cell function?
- Yes: 44% pre-debate → 57% post-debate (Dr. Greenbaum’s side)
- No: 16% → 19% (Dr. Herold’s side)I don’t know yet: 33% → 17%
- I don’t care: 6% → 7%
DEVELOPMENT OF AN ENCASPULATED CELL THERAPY FOR INSULIN-DEPENDENT DIABETES
Eugene Brandon, PhD (Director, Strategic Relations & Project Management, ViaCyte, San Diego, CA)
Dr. Eugene Brandon reaffirmed that ViaCyte is planning to begin a phase 1/2a trial of VC-01, the company’s beta cell progenitor encapsulation device for the treatment of type 1 and type 2 diabetes in 2014 – the same timeline the company gave at Biotech Showcase 2013 in January (for more on ViaCyte’s presentation at Biotech Showcase see page 77 of our JP Morgan Healthcare Conference 2013 full report at https://closeconcerns.box.com/s/rj1edd12eld3qorndbgl. VC-01 is comprised of a proprietary encapsulation device (called Encaptra) filled with beta cell progenitors (PEC-01) and alpha cells. The progenitor cells develop pancreatic functionality and encourage vascularization when implanted subcutaneously. In mice trials, PEC-01 developed into mature beta cells in about three months, a process that was associated with improvements in glucose levels. Once the cells were mature glucose levels normalized quickly whenever the mice’s glucose levels were artificially spiked (using streptozotocin [STZ]) with no evidence of hypoglycemic overshoot. ViaCyte had a pre-IND meeting the FDA last year, which Dr. Brandon characterized as being very positive. ViaCyte and the FDA agreed that the phase 1/2a trial will begin with one patient, to monitor VC-01’s safety. Assuming, the patient responds well, ViaCyte will gradually enroll additional patients and escalate the dose of PEC-01. At the 2013 Biotech Showcase ViaCyte CEO and President, Dr. Paul Laikind, stated that the trial will likely have a total enrollment of about 40 people.
ViaCyte plans to submit an IND for its beta cell progenitor encapsulation device, VC- 01, to the FDA in early 2014 and to initiate a phase 1/2a trial later in 2014. ViaCyte and the FDA agreed that the phase 1/2a trial will begin with one patient, to monitor VC-01’s safety. Assuming, the patient responds well, ViaCyte will gradually enroll additional patients and escalate the dose of PEC-01. ViaCyte then hopes to initiate a phase 2b/3 study in late 2016 or early 2017, which will last through 2019. It will then submit a biologics license application (BLA) in late 2019 or early 2020.
ViaCyte has had solid fundraising success lately for its VC-01 for the treatment of type 1 and type 2 diabetes. On February 13, JDRF and the California Institute for Regenerative Medicine (CIRM) each gave ViaCyte $3 million for VC-01. Earlier in December, the company was awarded another $3 million from CIRM for VC-01’s development and in October 2012 CIRM awarded ViaCyte $20 million.
In mice trials, PEC-01 developed into mature beta cells in about three months, a process that was associated with improvements in glucose levels. Vascularization begins to develop within the first four-weeks. Dr. Brandon detailed how the host tissue surrounding the VC-01 develops an extensive vasculature. ViaCyte’s data suggests that this vascularization is likely due to the secretion of growth and angiogenic factors by the PEC-01 cells within the Encaptra. When ViaCyte, implanted an empty Encaptra this vascularization did not occur. He explained that islets are so metabolically demanding they severely struggle to survive without a large oxygen supply. Fortunately, one of the unique properties of PEC-01s, according to Dr. Brandon, is that they survive the hypoxic period following implantation quite well and their secretion of factors to increase vascularization helps shorten the duration of this period.
Dr. Brandon stated that it is possible ViaCyte may need to make use a larger Encaptra in order to give people an adequate dose of PEC-01.
The PEC-01 cells are pancreatic progenitors with the potential to become beta cells, and are differentiated through a four-stage, ~two-week, in vitro protocol. Dr. Brandon stated that this protocol has been developed, refined, and optimized over about a decade and that it mimics the beta cell’s developmental process in the body. The differentiated cells can then by frozen such that they are stable for over ten months. It takes about two-to-four days for the cells to thaw, when they are needed.
ViaCyte’s proprietary encapsulation device, Encaptra, is based on a device Baxter was developing in the 1990s. Dr. Brandon highlighted that the Encaptra immunoisolates the beta cells, potentially limiting or eliminating the need for immunosuppressants. Encaptra is biocompatible and non-biodegradable. The device facilitates vascularization (the formation of blood vessels), likely increasing blood flow to the beta cells. Oxygen, nutrients, and proteins (such as insulin) are able to freely cross the Encaptra, however, it excludes host cells and retains grafted cells.
Dr. Brandon detailed how the VC-01 has multiple risk-reduction components. He explained that one of the key concerns people have with such a stem cell therapy is that if non- differentiated cells are implanted they might form a teratoma (a type of cancer made of cysts that contain one or more the three germ layers found in a developing embryo). Such teratomas are often non-malignant. ViaCyte’s’ strategies for reducing the risk of teratomas span its: manufacturing of the cells, cryopreservation of the cells, encapsulation of the cells, device loading system, subcutaneous implantation, and in vivo imaging.
Cell manufacturing: According to Dr. Brandon, PEC-01 is highly enriched for the desired product cells and no undifferentiated embryonic stem cells (which can become a teratoma) are detected in the product.
Cryopreservation of the cells: Dr. Brandon explained that by cryopreserving the cells, ViaCyte can perform quality control assays of the cells.
Encapsulation: ViaCyte has found that the Encaptra can contain a teratoma should one form (though Dr. Brandon repeatedly stated that ViaCyte has never had a teratoma form). Additionally, the Encaptra is retrievable making it easier to remove a teratoma within it.
Device loading system: ViaCyte is working to make loading cells into the Encaptra as simple for HCPs as possible. By doing so, ViaCyte can held ensure the viability of cells is maintained during loading. Additionally, it reduces the risk of damaging the Encaptra during loading or having cells enter the body on the outside of the device.
Subcutaneous implantation: By having the VC-01 implanted in the subcutaneous space, ViaCyte believes that implantation and explantation could occur with minimal surgical risk. Additionally, Dr. Brandon thinks that it minimizes the potential for localized toxicity effects and aids in the monitoring of inflammation.
In vivo imaging: It is possible to image the VC-01 using an ultra sound. Thus, one can monitor the device for unexpected cell growth.
The VC-01 is implanted in the subcutaneous space. ViaCyte has not determined the exact location of implantation yet, however, Dr. Brandon indicated it might be the lower back, flank, abdomen, or upper arm. He stated that ViaCyte probably want to avoid implanting the VC-01 in areas where people are likely to give themselves injections, to reduce the risk of a needle puncturing the Encaptra.
Notably, ViaCyte successfully performed continuous glucose monitoring on rats using the Dexcom seven. The CGM study determined that the rodents with VC-01 maintained normal blood glucose levels throughout the day and night for several days, despite having received streptozotocin (STZ; a glucose challenge).
- Dr. Brandon stated that we need a renewable source of islets that can be delivered without immunosuppression. The three key problems he identified with current islet transplants were the: 1) need for chronic immunosuppression, 2) transplant site complications, and 3) limited supply of pancreases. As a result of these challenges, only ~750 patients have successfully received islet transplants from human cadavers since 2000.
Questions and Answers
Dr. David Harlan: In your study where you administered STZ in the male and female mice, I was wondering if you have done similar studies in wild type mice?
A: The device protects from alloimmunity but not xenoreaction. We do not know about human alloimmunity, though an analogous device looked to be positive with this regards in humans. However, all of the antigens the mice would be exposed to from the human cells would drive xenoreaction. The device does not protect against this.
Q: Is the vascularization going into the cells?
A: It stays outside the membrane. It is a fairly open membrane from a protein perspective but the cells cannot get through. The cells inside the device cannot proliferate within a certain distance – we believe because of oxygen requirements.
Q: Isn’t that a problem?
A: Empirically that is the limit that we observe. Presumably the oxygen is a limiting factor for this proliferation. We are considering making devices that have a greater capacity. We may need something that is a little bigger in order to provide a large enough dose. So we are working on different strategies for shrinking the subcutaneous footprint in order to enable that.
Q: We saw exactly the same vascularization, however, in our panels there was almost no blood diffusion in those vessels.
A: That is interesting to think about – if this vascularization will be normal enough to provide an effective product. All I can say is that it appears to work.
Q: You did not show any C-peptide results, I was wondering if you have that kind of data.
A: I know others at the company have showed that C-peptide goes up. I did not bring that data with me.
Q: If you implant undifferentiated cells, is the strength of the device enough to withstand a teratoma? Do you really have a protection there in terms of safety?
A: We essentially put embryonic stem cells (ESCs) into the device and tried different levels of ESC contamination. We had an interesting finding – because of the oxygen limit the ESCs will form cartilage, presumably because cartilage is not oxygen demanding. We have not seen this break the device. We have seen it move the lumen out about a millimeter more, however, this does not cause the device to get to its burst pressure. We also can see that change in device size with an ultrasound.
Q: Does the device allow the passage of immunoglobulin?
A: The device is wide open to immunoglobulin. So that could be an issue when we move into clinic.
Q: Have you done hypoglycemia clamps? Stopping insulin production could be as important as starting insulin secretion.
A: That is a great suggestion; we have not done any clamping studies yet. That is certainly something we are considering. There are alpha cells in this device as well. The hope is that this community of endocrine cells will regulate glucose metabolism correctly.
Diabetes and the Gut Microbiome
STOPPING AUTOIMMUNE DISEASE IN TYPE 1 DIABETES: EXPLOITING THE GUT IMMUNE SYSTEM
Chantal Mathieu, MD, PhD (Katholieke Universiteit, Leuven, Belgium)
Dr. Chantal Mathieu’s excellent presentation provided a review of anti-CD3 trials and some of the key mistakes, as well as preclinical research on combination low-dose anti-CD3 and lactococcus lactis bacteria (secreting pro-insulin and IL-10). Combination therapy of anti-CD3 and lactococcus lactis induced stable, long-lasting diabetes reversal in 59% of recent onset diabetic NOD mice, far better than the individual components alone. Mechanistic studies indicate that the therapy works by the induction of Fox P3 Tregulatory cells. Dr. Mathieu wants to move the approach into the clinic and is working with ActoGeniX (the company that developed the special bacteria) to hopefully prepare a clinical trial in the next month.
Dr. Mathieu reviewed some of the key mistakes and learnings from trials testing anti-CD3. First, she noted that some of the earliest research into anti-CD3 demonstrated that there is a key window of opportunity for intervention. Contrary to what most would believe, earlier is not always better – in preclinical studies testing anti-CD3, mice that were four or eight weeks old had no benefit. Dr. Mathieu was also slightly critical of “big pharma,” which took teplizumab and otelixizumab over and “tried to repeat the studies in a typical phase 3 program” - e.g., multi-center trials with typical endpoints like A1c and glycemic control. In her view, these approaches are better kept for type 2 diabetes drugs or new insulins, and they are “not the way to go in type 1 diabetes” (rather, studies should use C-peptide). It is hard to know, of course, whether “big pharma” created the endpoints or whether FDA did; the agency is thought by some to be A1c-centric. GSK also “dramatically dropped the dose” in DEFEND, and in her view it was “no big surprise” that the trial was not successful. The lesson in all of this? “We should advise pharma even more than we already do,” she said. That, we suspect, would be welcome. She also highlighted that “everybody has a feeling that anti-CD3 is something that is worthwhile to go for.” We concur on this front and feel we have been hearing promising perspectives of late, although the failure of these two phase 3 trials was indeed a surprise and we can imagine it would be hard to find a partner to fund another major phase 3 trial, even if the confidence level and learning is significantly higher.
Dr. Mathieu reviewed preclinical work using a low dose of anti-CD3 and genetically modified lactococcus lactis secreting proinsulin and IL-10. The trial is aimed at inducing the reversal of autoimmune diabetes by restoring antigen specific tolerance. IL-10 is a disease modulating cytokine that pushes the immune system towards tolerance. A company called ActoGeniX succeeded in making bacteria that secretes IL-10 and proinsulin.
Newly diagnosed diabetic NOD mice were treated for five days with anti-CD3 plus six weeks of oral lactococcus lactis. The mice were then followed mice for eight weeks. Use of lactococcus lactis secreting proinsulin only had a small effect, reversing diabetes in 15% of the animals. Anti-CD3 alone reversed 25% of cases, combination anti-CD3 and lactococcus lactis secreting IL-10 reversed 40% of cases, and combination anti-CD3 with lactococcus lactis secreting proinsulin reversed 49% of cases. Most interesting was the combination of all three (anti-CD3 with lactococcus lactis secreting proinsulin and IL-10), which reverted diabetes in 59% of the mice. The treatment lasted eight weeks after stopping therapy and beyond. Mechanistic studies indicate the therapy works by the induction of Fox P3 Tregulatory cells. Dr. Mathieu wants to move the approach into the clinic and is working with ActoGeniX to hopefully prepare a clinical trial in the next month.
BACTERIA REFLUX CONTRIBUTING TO T1D
Olle Korsgren, MD, PhD (Uppsala University Hospital, Uppsala, Sweden)
In a fascinating lecture, Dr. Korsgren proposed that the first stage of type 1 diabetes may be an invasion of bacteria from the gut to the pancreas. The hypothesis is that these bacteria release toxins that provoke inflammation at focused areas of the pancreas – inflammation to which the beta cells are especially sensitive. This gradual, inflammatory process seems to occur to small degrees even in people without diabetes. However, as more and more areas of the pancreas become inflamed, autoimmune responses (and/or other factors) could arise in some individuals and thus induce type 1 diabetes. Dr. Korsgren’s model could explain why people with type 1 diabetes or type 1 autoantibodies have significantly smaller pancreases than the general population, even though the islets themselves have negligible mass. (This phenomenon is mysterious if type 1 diabetes is thought to be only a disease of beta-cell autoimmunity, but it makes sense if the islets are simply in the ‘line of fire’ of a progressive inflammation spreading destructively through the pancreas.) Much remains unknown and unproven about Dr. Korsgren’s hypothesis, but we were excited by his fresh perspective and eager to see how it might translate to preventive interventions.
Questions and Answers
Q: None of the evidence you presented dismisses the idea that autoimmunity is important. As you know, we don’t really understand what initiates loss of tolerance, and the pathway leading up to autoimmunity could be the one you describe.
A: This also encompasses the cytokine theory. I think we all agree.
Q: You may have mis-cited the literature when you spoke about a loss of induced pancreas volume. All we can say is that the pancreas is smaller in type 1 diabetes, and that it’s also smaller in prediabetes. Maybe the fact that you have a small pancreas predisposes you to those conditions.
A: I totally agree. I tried to show evidence of an ongoing inflammatory process, but of course we don’t know the real reason for the small size of the pancreas.
Q: You showed data from a model of progressive focal pancreatic inflammatory disease in rats. Did they eventually get diabetes?
A: The only thing we have so far is that T-cell response seems to include both CD8 and CD4. We have not seen an animal developing diabetes.
FECAL TRANSPLANT – BRIDGING T1D AND T2D
Max Nieuwdorp, MD, PhD (Academic Medical Center, Amsterdam, The Netherlands)
Dr. Max Nieuwdorp discussed the rationale for fecal transplants (FCT) and reviewed trial results. After citing some interesting facts about microbiota, he reviewed the general procedure for fecal transplant studies, emphasizing that strict criteria and screenings are used to select appropriate donors. The fecal sample is first mixed with saline and the patients’ GI tract is flushed to remove food before the infusion of the stool mixture. A recent study in the NEJM showed the ability of FCT to resolve recurrent C. difficile infection (which causes diarrhea and abdominal pain). In the study, the recipients showed increased fecal bacterial diversity similar to that in healthy donors, with the effect lasting up to six months (Van Nood, NEJM 2013). Dr. Nieuwdorp also reviewed several studies including a double-blind RCT (called FATLOSE; n=18) in obese males with metabolic syndrome, which found that participants receiving stool from lean donors exhibited improved peripheral and hepatic insulin sensitivity compared to those receiving their own stool samples. Dr. Nieuwdorp also discussed the potential for FCT in type 1 diabetes, as the microbiome in type 1 patients appear to differ from those in individuals without the disease. Current studies are evaluating whether FCT with healthy donor stool can inhibit the autoimmune attack and stabilize beta-cell function. In ending his presentation, Dr. Nieuwdorp remarked that while FCT is not a valid treatment modality, it is a valuable method for deriving novel probiotics.
Questions and Answers
Q: When you perform a fecal transplant, for how long do you modify the microbiome?
A: We see different effects. In some people, the effect lasts over six months. But that’s not the case in all patients. In some patients, the effect lasts three months, in some only two months, and in other patient groups six months.
Q: In other studies, have you repeated the procedure to establish a more permanent change?
A: That’s what we’re doing now. We’re doing repetitive fecal transplants in type 2 diabetes to see if we can change the host itself.
Q: How about viruses?
A: There’s a distinct interplay between viruses and the microbiome … These two keep each other at bay. I’m not sure if this will have an effect, and I will keep that in mind. I think that if you change the microbiome for a long period of time, you might change viruses as well.
Q: It’s been recommended that one should clean the bowel by lavage. What effect does that have?
A: The only answer I can give you is that in our NEJM paper, we included a lavage arm into the study. We saw no effect on the disease. The lavage flushes large amounts of food, but the bacterial colony is still there in the mucous layer. The fecal transplant starts a new state. Flushing itself doesn’t start new state.
Reversal of Autoimmunity and Transplantation Tolerance
MESENCHYMAL STEM CELLS FOR THE TREATMENT OF DIABETES
Anthony Pileggi, MD, PhD (University of Miami, FL)
Dr. Anthony Pileggi provided a cautiously optimistic overview of mesenchymal stem cells (MSCs), noting that “this field is really moving quickly.” Most interesting was his review of ClinicalTrials.gov studies of MSCs in diabetes, as well as a discussion of their potential in type 1 and type 2 diabetes. He believes MSCs offer new opportunities for treatment in diabetes, but there are also unaddressed scientific questions. He called for more mechanistic studies and research on stability, potency, and the safety of cellular products. Dr. Pileggi concluded that the data gathered to date “justifies cautious optimism.”
Research on MSCs has expanded exponentially in recent years. There are currently 306 trials of mesenchymal stem cells listed on ClinicalTrials.gov, with 102 in Asia, 73 in the EU, and 62 in the US (we were particularly surprised to see China’s disproportionate number). Around 25% of the trials are examining autologous (self) MSCs. Dr. Pileggi found 23 trials testing MSCs and diabetes (mostly in China), including eight in type 1 diabetes, four in type 2 diabetes, and six for complications (mostly ischemia) – we’re not sure why these three trial categories didn’t add up to 23.
The potential use of MSCs in diabetes includes immunomodulation (autoimmunity, islet organ transplantation), tissue repair, and metabolic function. Dr. Pileggi highlighted one study testing the safety and efficacy of MSCs in newly diagnosed type 1 diabetes in Brazil. The eight IV infusions of MSCs tempered the inflammation at new onset type 1 diabetes, “maybe extending the lifespan of beta cells.” In another study taking place at Uppsala University, patients with new onset type 1 diabetes are being treated with MSCs. Dr. Pileggi is excited to see the results, which are expected in May 2013 (ClinicalTrials.gov Identifier: NCT01068951). Preliminary results from a third study (ClinicalTrials.gov identifier: NCT01374854) suggest MSCs can reduce insulin requirements, A1c, fasting blood glucose, and increase fasting C-peptide.
In preclinical models of type 2 diabetes, MSCs have also shown a benefit. In a 2012 study appearing in Diabetes, infusion of MSCs ameliorated hyperglycemia in rats with type 2 diabetes. The improved metabolic control lasted for a couple weeks, and encouragingly, there was preservation of beta cell mass. Results suggested earlier treatment was better.
Dr. Pileggi posed some provocative questions and lingering issues: Are MSCs from patients with diabetes altered relative to those without diabetes? (Some studies suggest they are different.) Is the NOD model the best model of type 1 diabetes? What about issues surrounding oversight, standardization, cell products isolation, identification, trial protocols, and endpoints?
MULTIPOTENT MESENCHYMAL STROMA CELLS: POTENTIAL IN CLINICAL ISLET TRANSPLANTATION
Yoko Mullen, MD, PhD (City of Hope, Duarte, CA)
In a measured and meticulous lecture, Dr. Yoko Mullen explained that multipotent mesenchymal stroma cells can be a beneficial addition to islet transplants. Multipotent mesenchymal stroma cells – a type of adult stem cells found in bone, adipose, and other tissues – help to maintain cell homeostasis and regenerate damaged cells. Dr. Mullen’s group has conducted animal studies suggesting that when multipotent mesenchymal stroma cells are co-transplanted along with islets, they co-locate with the islets, promote revascularization, and exert immunomodulatory effects. Small preclinical experiments of co-transplantation have yielded high rates of diabetes remission. Given these results (and the that multipotent mesenchymal stroma cells are already used therapeutically in humans), Dr. Mullen is optimistic that MSC co-transplantation can improve outcomes in clinical islet transplantation.
A NOVEL ORAL VACCINE FOR TYPE 1 DIABETES BASED ON LIVE ATTENUATED SALMONELLA
Mohamed Husseiny, PhD (City of Hope, Duarte, CA)
Presenting preliminary experiments in mice, Dr. Mohamed Husseiny described a potential vaccine for type 1 diabetes based on oral delivery of live, attenuated salmonella bacteria. He explained that salmonella is taken up by the body’s antigen-presenting cells, where the bacteria multiply. (Outside of diabetes, salmonella has already been studied clinically as a vaccine, because the bacteria can serve as vehicles for plasmids – small bits of DNA that are then expressed inside the body.) Dr. Husseiny tested salmonella that were modified to carry plasmids of an autoantigen (preproinsulin), with or without plasmids for an immunomodulator (TGF-beta). In NOD mice, the salmonella-induced expression of preproinsulin alone did not significantly improve glycemia relative to the control. However, when the vaccine caused expression of both preproinsulin and TGF-beta, the mice’s symptoms improved significantly. In future studies of salmonella-based vaccines, Dr. Husseiny is enthusiastic to study other combinations of antigens and immunomodulators.
REVERSAL OF AUTOIMMUNITY VIA INDUCTION OF MIXED CHIMERISM UNDER A RADIATION-FREE ANTI-CD3/CD8 CONDITIONING
Defu Zeng, MD, PhD (City of Hope, Duarte, CA)
Dr. Defu Zeng presented data showing that when diabetic NOD mice underwent a radiation-free anti- CD3/CD8 conditioning regimen and then received bone marrow cells from healthy mice with a different MHC-type (the mouse version of HLA-type) their autoimmunity was reversed. These mice thereby had mixed chimerism induced (meaning they had both self- and host-immune cells). Mice with both recent- onset and late-stage diabetes were cured through this regimen. Though this regimen is still in very early research, there is some evidence that it could possibly work in humans; people with lupus who have undergone bone marrow transplants for leukemia have had their lupus cured. However, the classic bone marrow transplantation procedure is largely considered to be inapplicable for autoimmune diseases such as type 1 diabetes, because of the toxicity of the radiation or high-dose chemotherapy used for conditioning as well as the later potential for graft-versus-host disease.
Questions and Answers
Q: Thank you, that was a fascinating talk. At the Immune Tolerance Network we are very interested in mixed chimerism. In generally it requires very aggressive conditioning, though not necessarily radiation. Can you explain why anti-CD3/CD8 is sufficient when that has not been described by others?
A: After you inject the antibody, the antibody depletes part of the hosts’ T cells and also down regulates the T cell receptor making them disarmed. Before they come back you infuse donor T cells. The beauty of this regimen is that it allows a large dose of donor T cells.
Q: Is the dose of anti-CD3 similar to what has been used in new onset diabetes?
A: It is the same dose used in phase 3 trials of anti-Cd3 for type 1 diabetes.
Bart Roep, MD, PhD (Leiden University Medical Center, Leiden, The Netherlands); Defu Zeng, PhD (City of Hope, Duarte, CA); Samuel Strober, MD (Stanford University, Stanford, CA); Yoko Mullen, MD, PhD (City of Hope, Duarte, CA); Antonio Pileggi, MD, PhD (University of Miami, Miami, FL)
Q: Mixed chimerism induction and use of MSCs is a really exciting approach, especially in transplantation. In new onset type 1 diabetes, the challenge is finding an induction regimen that is acceptable. And possibly anti-CD3 may foot that bill. Complications are totally unacceptable in the context of type 1 diabetes.
Dr. Strober: A concomitant conceptual of mixed chimerism is you will not get graft versus host disease. If you can develop a regimen hat gives you stable mixed chimerism, the risk of graft vs. host disease is very low. That is what you’re trying to do.
Q: In the kidney transplant trial, I guess some of the patients had type 1 diabetes.
Dr. Strober: Yes.
Q: Did you look at autoimmunity before and after?
Dr. Strober: That was a conversation over dinner last night. We’ll try.
Q: In the pancreas transplant setting, you can get either a rejection or occurrence of autoimmunity. Could you speak to their relative importance?
A: Dr. David Harlan has a review article on this. The graft is rejected by both allo- and autoreactive cells. The auto-reactive T cells are more resistant to suppression.
Dr. Roep: Yes, but they are already activated, whereas the alloreactive response takes time to manifest. We might have to come back to this.
Scientific Achievement Award Recipient
EPIGENETIC MODIFICATIONS INDUCED BY INTERFERON-ALPHA IN HUMAN ISLETS: ROLE IN TYPE 1 DIABETES
Mihaela Stefan, MD (Mount Sinai School of Medicine, New York, NY)
Dr. Mihaela Stefan described how interferon-alpha (IFNa) is a critical cytokine in the development of type 1 diabetes and that it could be involved in genetic-epigenetic interactions that might trigger type 1 diabetes. Dr. Stefan has found that IFNa induces modifications of chromatin epigenetic marks (such as methylation and histone modification) in human pancreatic islets. In particular, the histone modifications IFNa induces are associated with increased mRNA expression of innate and adaptive immune response genes. Though the expression of these genes have positive antiviral and antiproliferative effects, they can also cause an autoimmunity and induce apoptosis. Thus, Dr. Stefan suggested that IFNa might be the missing link between viral infections and the development of type 1 diabetes.
Dr. Stefan began her presentation by describing past human evidence that IFNa is associated with the development of type 1 diabetes. For example, elevated mRNA IFNalevels were found in beta cells of type 1 diabetes patients. In a different study, elevated IFNa was found in the serum of people with type 1 diabetes and was correlated with enterovirus infection (one of the proposed triggers for the development of type 1 diabetes). Additionally, IFNa expression correlates with overexpression in islets of MHC class I antigens, which are important players in the type 1 diabetes autoimmune response.
Inflammation, Macrophages, and ER Stress
REGULATORY MYELOID CELLS MEDIATED IMMUNE SUPPRESSION VS. INFLAMMATION
Shu-Hsia Chen, PhD (Mount Sinai School of Medicine, New York, NY)
Dr. Shu-Hsai Chen explained that myeloid-derived suppressor cells (MDSC) are understood to be one of the central regulators of the immune system. MDSC can differentiate into either M1 or M2 cells. M1 cells tend to form in a type 1 cytokine environment, meaning that there are high levels of interferon (IFN)- gamma and IFN alpha. M1 cells produce proinflammatory cytokines and help mediate tumor cell killing and the inflammatory response. In contrast, MD2 cells forms in a type 2 cytokine environment, when there are high levels of interleukin (IL)-10, IL-4, and IL-13. M2 is known to produce IL-10 and Arg1, and to inhibit the inflammatory response. Dr. Chen continued to present preclinical research demonstrating that one can cause MDSC’s to differentiate into their M2 phenotype by controlling LILRB (PIRB in the mouse) signaling and that this can result control a diabetic mouse’s autoimmunity and prevent graft- versus-host disease.
Karin Bornfeldt, PhD (University of Washington, Seattle, WA); Janice Huss, PhD (City of Hope, Duarte, CA); Shu-Hsia Chen, PhD (Mount Sinai School of Medicine, New York, NY); Anthony Ferrante, MD, PhD (Columbia University, New York, NY); Feroz Papa, MD, PhD (University of California, San Francisco, San Francisco, CA)
Q: Does what we know about lipid storage diseases help us to find new directions to take this further?
Dr. Ferrante: There is only one known acid lipase. There are people who have the disorder. With the most severe form, people die very young. Their guts, liver, and bone marrow are full of lipids. The fascinating thing is that a group at Case Western were able to cure the mice by doing a bone marrow transplant. Several children have now been cured by doing these bone marrow transplants. I think that we will learn more about lipid metabolism.
Q: Could you discuss the role of MSDC in blood in humans?
Dr. Chen: In normal peripheral blood, we see it a lot only in cancer patients. MDSCs can mobilize from bone marrow. We can mobilize MDSCs and then prevent GvHD in a mouse model. We can also identify a significant population and induce T-regulatory cells in humans.
Q: On the other side is there any evidence of loss of MSDCs leading to autoimmune disease?
Dr. Chen: That is a very interesting question. If you have an autoimmune disease you have inflammation. Those people probably have the M1 phenotype, which causes inflammation. We are hoping that we can move it into the M2 phenotype.
EPIGENETIC MECHANISMS AND BETA CELL BIOLOGY
Anil Bhushan, PhD (University of California Los Angeles, Los Angeles, CA)
In a talk focused on basic science, Dr. Anil Bhushan discussed the decline in beta cell replication and expansion that accompanies aging. Studies show that the expression of p16Ink4a is indicative of the regenerative capacity of beta cells, with higher p16 levels associated with lower regeneration. The expression of p16 depends on levels of Ezh2 – younger cells have higher levels of Ezh2 and do not express p16 while aging cells have decreased Ezh2 levels and higher levels of the protein MLL that promotes p16 transcription. Dr. Bhushan detailed studies that investigated whether replenishing Ezh2 can restore regeneration capacity in aged islet cells. These studies indicated that the overexpression of Ezh2 coupled with the loss of MLL repressed p16 expression and increased beta cell proliferation in aged islets.
Human Pancreas Development
A CAREFUL LOOK AT HUMAN ENDOCRINE PANCREAS DEVELOPMENT
Marcela Brissova, PhD (Vanderbilt University, Nashville, TN)
On Saturday morning, Dr. Marcela Brissova detailed differences in the development of the human and mouse pancreas, to a smaller audience than in the conference’s prior days (about 60 people). One of the central differences he explained is that endocrine cell clusters form throughout the human pancreas while in mice they form in cohorts around the central region of the pancreas. Additionally, mice and human pancreases differ greatly in the ratio of different hormones they produce; the human pancreas produces ~30% insulin, ~30% glucagon, and ~30% somatostain, whereas the mouse pancreas produces ~55% insulin, ~40% glucagon, and ~5% somatostain. She explained that understanding the development of the human pancreas could help researchers devise new sources of islets for transplantation from stem cells. Additionally, an improved understanding of human pancreas development could guide researchers on how to increase beta cell regeneration or perform genetic reprogramming of beta and alpha cells.
Dr. Brissova described how human islet function and function differs across a person’s lifespan. She noted that beta cell proliferation tends to be greatest during the neonatal period and that the level of beta cell proliferation throughout life is highly heterogeneous between people.
DEVELOPMENT OF AUTHENTIC HUMAN BETA CELL LINES
Philippe Ravassard, MD (ICM-INSERM, Paris, France)
Dr. Philippe Ravassard discussed his group’s latest progress in developing authentic human beta cell lines that can be used for preclinical research. Previously, Dr. Ravassard and colleagues have demonstrated a procedure for: collecting human pancreas progenitor cells, transducing them with genes that promote ongoing proliferation, transplanting them into SCID mice, letting them develop into insulin-producing cells, and then amplifying them into cell lines through successive cycles of transplantation and in vitro cultivation. Though these transgenic cells were genetically and phenotypically quite similar to natural human beta cells, they were abnormal in that they never stopped proliferating in the body. To address this issue, Dr. Ravassard’s group recently developed cells from which the eternal-proliferation genes can be excised at will. Early studies suggest that the excision process works fairly well, turning the vast majority of proliferative cells into “authentic” human beta cells. So far the researchers have produced 35 cell lines from 7 independent human fetal pancreases. Dr. Ravassard believes that these cell lines are ready to be used in research on drug discovery and preclinical models of cell transplantation. He also looks forward to refining the excision process, so that the eternally proliferating cells can be more thoroughly converted to normal beta cells.
Diabetes Care and Drugs (Non-Cure Therapies)
Clinical Trial Controversies in Diabetes Care
DCCT/EDIC RESEARCH GROUP UPDATE
John Lachin, ScD (George Washington University, Washington, DC)
Dr. John Lachin provided a broad overview of the design and results of the DCCT/EDIC studies, highlighting a few key points: 1) DCCT differences in A1c consistently explain 90-99% of the differential risk of complications between the conventional and intensive groups; 2) hypoglycemia is a “failure of the technology to implement intensive insulin therapy”; 3) the observed “metabolic memory” effect may be explained through hyperglycemia-induced oxidation or epigenetics, though further study is required; and 4) the degree of follow-up in the DCCT/EDIC “has been remarkable.” Overall, he made a very strong case that levels of glucose drive complications, and this effect is sustained over a very long period of time. The success in learnings from DCCT/EDIC make us even more eager for a “modern-day” DCCT where continuous glucose monitoring is used.
DCCT differences in A1c between the intensive and conventional groups consistently explain nearly all of the differential risk of complications. Dr. Lachin repeatedly emphasized this fact, one that has held up over time. At the end of the DCCT, 96-99% of the difference between the treatment groups in incidence of complications was explained by levels of A1c. The same has been observed throughout EDIC. Dr. Lachin also reviewed the data on cardiovascular disease outcomes (NEJM 2005), and again, 97% of the long-term effect on incidence of cardiovascular disease is explained by DCCT differences in A1c (compared to ~40- 50% for other markers like microalbuminuria). We heard, of course, the oft-stated statistic coming out of DCCT - for every 10% reduction in A1c points, there was a 43% reduction in the risk of retinopathy progression.
Hypoglycemia in the DCCT represented “a failure of the technology to implement intensive insulin therapy.” Dr. Lachin discussed the high rate of severe hypoglycemia in the intensive group (16 per 100 patient years) compared to the conventional group (five per 100 patient years). In his view, this is a reflection of the technology used at the time to implement tight glycemic control – this is, of course, also related to patients using NPH and regular insulins in DCCT vs. much more stable analogs today that are used by many patients. He did not elaborate on specific weaknesses in particular technologies or therapies. Happily for patients, when the DCCT rate of hypoglycemia is compared to more recent rates in STAR-3, the JDRF CGM trial, and the T1D Exchange, it’s always clear that things have improved substantially.
The observed “metabolic memory” effect may be explained through hyperglycemia- induced oxidation or epigenetics, though further study is required. Dr. Lachin discussed an ancillary study of skin collagens and advanced glycation end products (Diabetes 1999). In the 216 patients studied, AGEs had a strong association with the risk of microvascular complications during the DCCT, a finding that was independent of A1c. When adjusting for the level of skin collagens, 95% of the DCCT’s A1c effect is actually explained by skin collagens. In his view, this suggests A1c affects skin collagens, which correspondingly affect outcomes. Dr. Lachin also discussed a small pilot study being jointly conducted with City of Hope researchers.
“The completeness of follow-up has been remarkable.” Dr. Lachin emphasized that DCCT/EDIC have “very little missing data,” which makes the results “highly definitive.” At the start of EDIC, 96% of the original DCCT cohort was enrolled. After 11 years of follow-up, 93% of the original DCCT cohort was enrolled in EDIC (96% of surviving patients). Incredibly, even in 2012 (18 years of follow-up), 88% of the original DCCT cohort is still enrolled. We’re sure this level of retention is not easy to achieve, though its incredible to see and a testament to the hard work of the DCCT/EDIC research team.
“The modern day course of diabetes has been tremendously affected by the population level declines in A1c.” Dr. Lachin reviewed a 2009 paper (Nathan et al., Arch Int Med) that compared the incidence of severe complications in DCCT/EDIC to the Pittsburgh Epidemiology of Diabetes Complications study. The incidences of proliferative retinopathy, nephropathy, and cardiovascular disease were 50%, 25%, and 14%, respectively, in the DCCT conventional treatment group, similar to the 47%, 17%, and 14% incidences in the EDC cohort. By contrast, the DCCT intensive therapy group had substantially lower cumulative incidences (21%, 9%, and 9%) and less than 1% became blind, required kidney replacement, or had an amputation because of diabetes during that time – certainly, much more welcome outcomes and impressive for that era.
GLUCOSE VARIABILITY: DOES IT MATTER?
Robert Vigersky, MD (Walter Reed National Military Medical Center, Washington, DC)
Dr. Robert Vigersky’s talk had a single aim: to convince the audience that glycemic variability matters. He did just that by addressing the theoretical basis for glycemic variability’s effect on outcomes (oxidative stress, inflammation, growth factors), reviewing the link between glycemic variability and outcomes (a slew of studies in many areas that are correlative), and discussing normal glycemic variability in people without diabetes (new evidence linking higher BMIs with higher glycemic variability). Notably, Dr. Vigersky believes glycemic variability may even be a more sensitive metric for predicting the onset of diabetes in those who are at risk. There’s no question that data is certainly emerging on a number of fronts, though there is nothing definitive at this point – we hope Dr. Irl Hirsch’s FLAT-SUGAR pilot study will lead to a long-term outcomes trial.
Glycemic variability has been linked to inflammation, oxidative stress, and growth factors, forming the theoretical basis for its effect on outcomes. Dr. Vigersky quickly flashed Dr. Michael Brownlee’s hypothesis on screen: a complicated series of biochemical pathways linking higher glucose variability to oxidative stress. He also discussed in vitro data demonstrating the adverse impact of glucose variability. Work from Ceriello et al., found that cells exposed to variable glycemic environments experience more oxidative stress and more apoptosis relative to high glucose alone (Quagliaro et al., Diabetes 2003). In another study comparing cell culture environments with high glucose and cultures with high glycemic variability (alternatinghigh and low), more VEGF was produced in the high glycemic variability culture (Sun et al., Mol Cell Biochem 2010). In 2006, Monnier published his paper in JAMA linking MAGE in type 2 diabetes patients to a urinary marker of oxidative stress. His findings were not confirmed in a similar 2008 Diabetologia study; however, Dr. Vigersky emphasized that the later study used a different methodology and studied type 1 patients (and used technology that wasn’t really reliable for assessing the impact of glycemic variability).
While there are no prospective studies linking higher glycemic variability to worse clinical outcomes, there is substantial correlational evidence. Dr. Vigersky cautioned that none of the studies provide causation, but he hopes that they “pique interest.” In his view (and we certainly agree), investigators should be measuring and following glycemic variability to better understand why some patients have worse clinical outcomes. We are also glad that current monitoring technology that can finally show the degree to which glycemic variability matters: historically, there has not been accurate, reliable CGM that patients and HCPs could use in trials and we’re glad to see that has changed so that this question can be reliably assessed. Dr. Vigersky ran through a large array of studies:
Postprandial studies – Decode, STOP-NIDDM, NAVIGATOR: The Decode study (Lancet 1999) found that for every level of fasting glucose, there was an increased hazard ratio for death as two-hour postprandial glucose increased. The STOP-NIDDM trial found that use of acarbose to prevent postprandial excursions had a positive effect on cardiovascular disease. The NAVIGATOR study was a counterpoint, finding that nateglinide to prevent postprandial excursions was not effective in improving cardiovascular events or mortality.
Studies are emerging that link glycemic variability to atherosclerosis: A Chinese study in 216 type 2 patients found that glycemic variability (MAGE) was associated with subclinical atherosclerosis as measured by carotid intima media thickness (CIMT). Another study found that at every level of A1c, the higher one’s postprandial glucose, the higher his or her CIMT. Said Dr. Vigersky, “these two studies suggest a relationship between glycemic variability and the burden of atherosclerosis.”
A growing body of evidence in the hospital has linked glycemic variability with adverse outcomes: A 2008 study (Krinsley et al., Crit Care Med) found that glycemic variability was a strong independent predictor of mortality in critically ill patients. The data was consistent regardless of mean glucose during the ICU stay – even in patients with a mean glucose of 70-99 mg/dl during their admission, linear increases in mortality were seen as glycemic variability rose. A 2010 study (Hermanides et al., J Crit Care Med) in an impressive ~6,000 patients similarly found that higher glycemic variability (as measured by MAG, a time-related measure of glycemic variability) was linked with higher ICU Mortality. A 2011 Su et al., study appearing in Cardiovasc Diabetol found that intraday glycemic variability was associated with the presence and severity of coronary artery disease in patients with type 2 diabetes. And a very recent 2013 study from the same group (Su et al., Diabetes Care) found that high glycemic variability (MAGE) at hospital admission was significantly associated with incidence of major adverse cardiovascular events at one year. Glycemic variability was more important than admission glucose and prior long-term abnormal glycometabolic status in predicting one-year MACE.
Dr. Vigersky also discussed what “normal” glycemic variability is in people without diabetes. He cautioned that there is “quite little data” on this topic, though it is important forcomparison purposes. A 2010 JDRF study (Diabetes Care) represents the best study on this topic, and maybe the only study in a normal weight population without diabetes. The study found that glucose values ≤60 and >140 mg/dl are uncommon in healthy individuals without diabetes. There have also been several studies evaluating glycemic variability in obese individuals. It would be valuable to have this study done again with more accurate, reliable, and easier-to-use CGM. A 2011 study (Ma et al., Obesity) found that glycemic variability was higher in abdominally obese individuals relative to non-abdominally obese individuals – this seems logical since pre-diabetes is likely higher in overweight individuals. Dr. Vigersky got the audience’s attention with a review of CGM data from individuals appearing on Season 10 of The Biggest Loser television show. Relative to non-obese individuals, the obese patients had higher glycemic variability; their BMIs were also “strongly” correlated with standard deviation of blood glucose (R2=0.7).
Dr. Vigersky concluded that the clinical significance of glycemic variability is unknown, but it may be an important risk factor and marker for: microvascular complications, macrovascular complications, hospital mortality, and predicting future hypoglycemia. He believes that glycemic variability may even be a more sensitive metric for predicting the onset of diabetes in those who are at risk – this would be quite a compelling proposition for early intermittent use of CGM and we hope studies could be conducted on this front.
CONTROVERSIES IN THE ACCORD TRIAL: CLARITY, CONFUSION, OR CONFIRMATION
David Kendall, MD (Eli Lilly, Indianapolis, IN)
Dr. Kendall provided a broad review of landmark clinical trials, focusing on the results and implications of ACCORD. He began by providing a historical perspective, noting that the rationale for ACCORD stemmed from remaining questions about glycemic control and macrovascular complications. ACCORD, however, provided a “real conundrum” as investigators noticed an unanticipated increase in all-cause mortality. Dr. Kendall noted that among the landmark trials, ACCORD represents an outlier – it is reasonable to suppose that there were unique characteristics of the trial and/or patient population that require further questioning; however, Dr. Kendall concludes, “we should not throw the proverbial glycemic control baby out with the bathwater.” Turning to clinical implications, Dr. Kendall noted that ACCORD identified higher-risk patients who may not benefit from intensive glycemic control: patients with longstanding poor glycemic control (suggesting a history of not responding to therapy), patients with a history of severe hypoglycemia independent of their treatment targets or regimen, and patients that do not respond to aggressive therapy yet continue to intensify treatment. In general, HCPs should look out for severe hypoglycemia and divergence from glycemic targets, as these red flags are associated with higher mortality risk.
Dr. Kendall provided a historical perspective, noting that current glycemic targets are based on decades of clinical trials starting with UGDP and including UKPDS, DCCT, EDIC, ACCORD, ADVANCE, and VADT. This history has allowed researchers to evaluate previous epidemiological-based observations that higher blood glucose exposure increases the risk of complications. UKPDS and DCCT demonstrated that the risk of microvascular complications increases with A1c in an exponential fashion, and that decreasing blood glucose levels reduced this risk. ACCORD did not attempt to reaffirm this association and instead examined the relationship between glucose and major cardiovascular complications – epidemiological data had indicated that the risk of macrovascular complications increases with A1c in a relatively linear fashion. This more linear relationship – compared to the exponential curve for microvascularcomplications – suggested that macrovascular complications may depend less on glucose alone and that the potential benefit of glucose lowering may be smaller.
Dr. Kendall broadly reviewed the study population and results of ACCORD, ADVANCE, and VADT. He noted that ACCORD and ADVNACE look similar in many respects, as they both recruited high-risk patients with type 2 diabetes who had similar durations of disease, most with a prior history of cardiovascular disease. However, the average baseline A1c of the study populations were different, with those in ADVANCE having A1c levels that represented patients early in the disease progression, those in ACCORD representing patients roughly mid- way through the disease, and those in VADT with higher A1c levels approaching the need for insulin. While each trial showed that intensive glycemic control trended toward reducing macrovascular complications, none demonstrated a signification reduction in cardiovascular events.
Pointing to the “real conundrum” in ACCORD, Dr. Kendall discussed the unanticipated increase in all-cause mortality observed in the intensely treated participants. Dr. Kendall reflected on the media’s reaction to this result, noting that people initially concluded that intensive therapy was dangerous rather than beneficial. Dr. Kendall remarked that he – along with other academics – had begun to question whether A1c targets needed to be increased and whether HCPs should favor monotherapy over combination therapy. In general, the results first prompted a heightened reaction “not based on careful assessment of the data.”
Dr. Kendall then provided high-level takeaways from the landmark clinical trials. DCCT and UKPDS provided the initial evaluations of glycemic control and complications in patients with type 1 or type 2 diabetes. The subsequent ACCORD, ADVANCE, and VADT trials investigated whether further intensifying glycemic control could improve macrovascular complications in patients at different disease stages. While every trial demonstrated that glycemic control decreased the risk of microvascular complications, only the follow-up studies for UKPDS and DCCT showed a similar finding for macrovascular complications. Dr. Kendal remarked that he is personally convinced that researchers “never need to do another study to confirm or support that glycemic control reduces microvascular complication risk in diabetes.” However, while evidence suggests that early intervention may later reduce the risk of macrovascular complications, the effects of therapy in the short-term remains unclear. Furthermore, researchers still need greater clarity on the question of intensive therapy and mortality.
Turning to clinical implications, Dr. Kendall identified high-risk patients who may not benefit from intensive glycemic control. Contrary to initial assumptions, higher rates of mortality in ACCORD were not correlated with age, insulin use, or attempting to achieve a lower A1c level. The trial showed that mortality risk may increase with duration of diabetes, but this trend was not conclusive. Those at highest risk were actually patients with longstanding poor glycemic control (suggesting a history of not responding to therapy), patients with a history of severe hypoglycemia independent of their treatment targets or regimen, and patients who did not respond to aggressive therapy yet continued to intensify treatment. Neither intensive glycemic control itself nor the speed of lowering blood glucose appeared to predict an increase in mortality risk.
Dr. Kendal noted that severe hypoglycemic increased the risk of mortality in both the intensive and standard treatment groups. However, having one or more events of severe hypoglycemia was associated with a greater increase in mortality risk in the standard therapy group.
To conclude his presentation, Dr. Kendall briefly discussed the blood pressure and lipid subtrials of ACCORD. ACCORD showed that targeting a lower systolic blood pressure (difference of ~20 mmHg lower than the standard group) did not further reduce the risk for cardiovascular disease, except perhaps in people with a history of stroke or other CV events. ACCORD also demonstrated that adding a lipid-lowering medication (fenofibrate in this case) on top of statin therapy provided no added benefit in terms of cardiovascular risk reduction.
USE OF BARIATRIC SURGERY TO CURE DIABETES
Philip Schauer, MD (Cleveland Clinic, Cleveland, OH)
Giving the second-to-last presentation of the conference, on a Saturday, Dr Philip Schauer described to an audience of ~50 people, how bariatric surgery (which Dr. Schauer likes to also call metabolic surgery due to its effects beyond weight loss) can cause long-term remission of type 2 diabetes in some patients. As evidence, he detailed three randomized control trials comparing various bariatric/metabolic surgery procedures against conventional diabetes treatment, which found that these surgeries were associated with strikingly high rates of remission, whereas conventional pharmacotherapy was linked to little-to-no cases of remission. Dr. Schauer highlighted that only 19% of people with type 2 diabetes are at goal for A1c (<7%), blood pressure (<130/80), and LDL (<100 mg/dl). Given this poor rate, he argued that it might be time to consider an alterative to polypharmacy.
Dr. Schauer reviewed three randomized control trials (RCT), demonstrating that bariatric/metabolic surgeries are associated with more type 2 diabetes remissions than pharmacotherapy.
The first randomized controlled trial (RCT) Dr. Schauer detailed, found that gastric bypass caused remission in 73% of people with early diabetes not on insulin (<2 years; mean A1c=7.7%; BMI: 30-40 kg/m2), whereas only 13% of people on convention treatment experienced a remission.
The second trial (n=60), compared gastric bypass, biliopancreatic diversion (BPD) and conventional pharmacotherapy, in people with type 2 diabetes (mean A1c=8.5%; mean BMI=45 [range: 35-50]). The trial found that nobody on conventional therapy experienced a remission (defined as an A1c<6.5%) in their diabetes, while 95% with BPD and 75% with gastric bypass reached remission.
The third study, STAMPEDE, was a head-to-head trial of roux-en-y gastric bypass (RYGB) and intensive medical therapy in obese people with diabetes (Schauer et al., NEJM 2012). STAMPEDE found that bariatric surgery was more effective than intensive medical therapy in achieving glycemic control (defined as an A1c ≤6%). In the trial, 42% of people undergoing RYGB (n=50) and 37% of those receiving a gastric sleeve (n=49) achieved an A1c ≤6% after one year, whereas only 12% of those on medical therapy (n=41) achieved an A1c ≤6%. Additionally, 100% of the RYGB patients and 73% of gastric sleeve patients who had an A1c ≤6% did so without the use of medications. Additionally, people who underwent either an RYGB or a gastric sleeve saw significantly greater improvements in their cardiovascular risk factors. For example, medical therapy was associated with an 11% increase in HDL, while RYGB and gastric sleeve was associated with a 28% increase (p=0.001). Furthermore, bariatric surgery patients accomplished this while reducing the number of cardiovascular medications they were taking. For ourfull coverage of the STAMPEDE trial, see our ACC 2012 report at http://www.closeconcerns.com/knowledgebase/r/5dd2ddad.
Bariatric surgery has been found to improve microvascular complications. In a study of 52 patients who had type 2 diabetes for a mean of 8.6 years (baseline BMI=49 kg/m2, A1c=7.7%), bariatric surgery (69% RYGB) caused nephropathy remission in 58% of patients. Patients’ baseline micro-macroalbuminuria on average was 38% (Heneghan, et al., SOARD, 2012). Dr. Schauer stated that similar results have also been seen with BPD, though he did not detail these.
Dr. Schauer described several non-randomized trials of bariatric surgery, highlighting improvements in cancer rates, cardiovascular outcomes, and type 2 diabetes incidence.
The Swedish Obese Subjects (SOS) study (Sjöström et al., NEJM 2007) enrolled 2,010 adults who underwent bariatric surgery (68% underwent vertical-banded gastroplasty) and 2,037 matched controls in a non-randomized prospective fashion. After 20 years follow up, the number of first-time cancers was lower in the bariatric surgery arm (n=117 or 6% of the group) than in the control (n=169 or 8% of the group). The hazard ratio for developing a first-time cancer after undergoing bariatric surgery was 0.67 (95% CI=0.53-0.85).
Additionally, an analysis of the people with type 2 diabetes in the SOS study (n=345 for the surgery group and n=262 for the control) found that bariatric surgery significantly reduced the incidence of cardiovascular events (HR=0.63; 95% CI: 0.45- 0.90). More specifically, bariatric surgery was associated with significantly fewer myocardial infarctions over the 20 years of follow up (HR=0.59; 95% CI: 0.38-0.92).
In a Utah-based prospective study (Adams et al., JAMA 2012), included 418 RYGB patients, 417 people who sought but did not have surgery (control group one), and 321 randomly selected people from a population-based sample not seeking weight loss surgery (control group two). The incidence of type 2 diabetes was reduced in the RYGB group from 17% to 2%, with 62% of people with diabetes experiencing remission. In contrast, only 8% of control group one and 6% of control group two were in remission after six years, and the incidence of type 2 diabetes in these groups was 17% and 15%, respectively (baseline type 2 diabetes rates not provided). Additionally, 42% of the RYGB arm (vs. 18% of control one and 9% of control two) were in remission for hypertension and only 16% still had hypertension at follow up. In contrast, 31% of control one and 33% of control two had hypertension.
Dr. Schauer concluded by nothing that while weight loss associated with bariatric surgery is an important driver of metabolic improvements, changes in incretin levels also appear to play a key role. In one study gastric bypass patients had near normal glucose tolerance after one and two years of follow up. On average these patients experienced a 5.8-fold increase in overall pancreatic beta cell function. Both gastric bypass and sleeve gastrectomy were found to stimulate incretin secretion including markedly increased levels of postprandial GLP-1. While these two bariatric surgery procedures caused similar levels of weight loss, gastric bypass was associated with greater improvements in insulin sensitivity and a larger reduction in truncal fat.
John Lachin, ScD (The George Washington University, Rockville, MD); Philip Schauer, MD (Cleveland Clinic, Cleveland, OH); Robert Vigersky, MD (Walter Reed National Military Medical Center, Washington, DC)
Q: Knowing what is available now about glycemic variability, particular mortality in hospitalized patients, do you have any favorite agents that actually have more impact on glycemic variability?
Dr. Vigersky: I think many clinicians in the room have been very impressed with the GLP-1 analogs. They address a triple threat: they cause weight reduction, they reduce glycemic variability, and they improve overall glycemic control. We don’t have long-term outcomes data to see which, if any of these, is going to improve things. But I think the incretin approach, as well as acarbose, is one that will reduce glycemic variability.
Q: Does anybody use alpha-glucosidase inhibitors anymore?
Dr. Vigersky: We don’t, but it is very popular in Germany and Asia. I think that there is a cultural issue with use of that drug. I will not say more; it also is cheap. While it generally is not used here it is used in other places.
Q: John, you gave a great overview of the DCCT. Can you comment on the 11 deaths that occurred during the DCCT? And since some of those patients had diabetes for 15 years, I wonder if you saw some of the effects seen in ACCORD – more problems in people in tight glycemic control who entered the study with longer duration disease.
Dr. Lachin: In terms of the 11 subjects, I cannot recall. We published a paper on adverse events in the DCCT. It described the risk of hypoglycemia and other adverse outcomes. All the causes of death were listed. I cannot recall a pattern. Regarding an ACCORD-like phenomenon, we really have not seen anything that would approach that. We’re only now beginning to do an analysis of mortality. I cannot comment on that. But we’re in the process of developing a paper on that.
Q: For me, one of the most important observations in the last ten years is metabolic memory. It is so interesting and clinically it is unbelievably important. So I guess my question is you did so much statistical analysis when the trial first ended to understand what are the correlations with these outcomes and you told us it is 90% glucose. Is there any sort of statistical observation on who goes into legacy, who does not go into legacy?
Dr. Lachin: Unfortunately, I was only able to focus on what explained the difference. In the cohort it is the total level of A1c over the total duration that determines the outcome. That applies to both the conventional and the intensive group. It still does not explain all of the risk. One of the biggest missing links is the pre-DCCT exposure. They were on average running an A1c of 8-9% for many years, and that will have a profound effect on their progression. In general a person’s glycemic control ten years ago will have a greater impact on their progression than their A1c six months ago. However, improving a person’s glycemic control now certainly is much better than doing nothing.
Q: There was a group in the DCCT intensive cohort that didn’t do well – they had higher A1cs than the average. For the intensive group that didn’t achieve a better A1c, did they show a memory effect?
Dr. Lachin: I cannot say we’ve done that analysis. I don’t believe we did an analysis that was stratified by A1c within each group. We’ve only done the group as a whole. That’s a good question.
Q: Some people have said and there was data published in ’95, showing that for the same A1c there were differences in the control and intensive group’s outcomes that could be due to glycemic variability. As I understand it, that statistical analysis took into account the improvement but not the absolute value of someone in the control group versus the intensive group. The speculation is that people in the intensive group were on pumps that would limit glycemic variability.
Dr. Lachin: In my paper there was a figure that for a given level of A1c a subject in the control group had a higher risk than a subject in the intensive group. I am here to confess that I made a mistake in how I used a model to generate that figure. That analysis was flawed. Later that figure was used as evidence for the importance of glycemic variability. We published a follow-up paper in Diabetes in 2008; in that paper what we did was we stratified subjects by their actual A1c and we showed that there really was no difference in their risk.
Q: For patients with type 2 diabetes, we’ve tested the hypothesis that if you tell somebody what’s good for their health, they will do it. Our surgical colleagues will ask why we aren’t referring our patients. I tell them that we are, but patients just don’t want to take that step. Have you found any ways at the Cleveland Clinic that make this therapeutic option more attractive?
Dr. Schauer: In folks with more severe obesity, diabetes brings hypertension, dyslipidemia, sleep apnea, orthopedic problems, and they are virtually unemployable. It’s an easier sell in those patients. In the STAMPEDE trial, we took folks as low as 27 kg/m2. It was hard to get folks in that range. They were patients with more advanced type 2 diabetes.
Q: But that’s not reimburseable at 27 kg/m2…
Dr. Schauer: It depends on where you are. At the Cleveland Clinic, the health plan lowered the BMI criteria to 30 kg/m2 with diabetes that is not well controlled with medical therapy. You have to talk to the patients somberly about the consequences of poorly controlled diabetes and the risk of complications. It’s also important for physicians and endocrinologists to share the real risks of surgery. But the risk is similar to operations like appendectomy in the hands of an experienced surgeon.
Q: I was wondering if you could comment on the link between GLP-1 and pancreatic cancer.
Dr. Schauer: No, we looked at this in bariatric surgery – bariatric surgery is associated with increases in GLP-1 levels. The SOS study showed that the surgical group had a dramatic decrease in cancers, particularly among women. Pancreatic cancer was reduced.
Dr. Vigersky: There have been two recent events related to that in recent weeks. One was a paper showing a doubling of the rate of pancreatitis of those using incretins. Then just two days ago the FDA said that they had received a number of reports about pancreatic caner in people taking incretins. The recent database analysis has all the limitations of database analysis. Some of those limitations are troubling because even though you show an absolute decrease in risk, it is all based on coding. It is only reliable if the coding is accurate. So in that study they said they captured the codes for obesity and alcoholism, which both have an increased risk of pancreatitis. The problem is, how many of you code your patient as obese if they have a BMI between 30 and 35? My experience is that people are coded for their comorbidities not for obesity. Payors tend to not pay for obesity and people are not very good about coding what they are not paid for. The same issue exists for alcoholism. So I think that we have to be very cautious about how we draw conclusions from these database analysis. That said, surely that group has interesting histological data showing that there are changes in the pancreatic ducts that could be worrisome. None of the groups that have looked at this prospectively, have found an increased risk. The decision to prescribe these or not should not be influenced by this data at this time.
Debate: The Beta Cell Will Be Preserved with Currently Available Therapy in Type 2 Diabetes
Philip Raskin, MD (University of Texas Southwestern Medical Center, Dallas, TX)
Dr. Philip Raskin argued that type 2 diabetes is a disease of progressive beta-cell failure caused by hyperglycemia, and that early initiation of insulin can ameliorate glucotoxicity, thereby helping to preserve the beta cell. He suggested that GLP-1 agonists might also have protective effects on beta-cell function but said that well-designed studies on this question have not been carried out.
“Type 2 diabetes is a disease of progressive beta-cell failure caused, in my opinion, by hyperglycemia.” Dr. Raskin mentioned impaired insulin action and glucagon excess as two other main problems in type 2 diabetes. (He did not describe basal hyperinsulinemia as a problem per se, but rather as the body’s inadequate effort to compensate for decreased insulin sensitivity.) Progressive loss of beta-cell function and/or volume have been seen in many studies, with various treatments (Butler et al., Diabetes 2003; UKPDS, Diabetes 1995). Thus, Dr. Raskin remarked that sulfonylureas’ lack of long-term efficacy is not due to failure of the sulfonylureas, but rather failure of the beta cells. He also argued that glucotoxicity is the main mediator of beta-cell failure. To support this view, Dr. Raskin cited a mouse study in which the survival of syngeneically transplanted islets was even worse under hyperglycemic conditions than normoglycemic conditions (Biarnes et al., Diabetes 2002).
“Aggressive initial treatment with insulin helps!!!” Dr. Raskin cited two randomized controlled trials in which early insulin therapy had positive, year-plus-long effects on insulin response and glucose levels. One trial, conducted in nine centers in China (n=382), assessed whether patients kept their diabetes in remission for a year without medications. This high bar was met by 45-51% of patients given insulin therapy but only 27% of those randomized to oral drugs. In another study, conducted by Dr. Raskin’s group, 63 treatment-naïve, newly diagnosed type 2 diabetes patients were all given premixed insulin aspart and metformin for three months, after which they were randomized either to continue on insulin or to switch to oral drugs. Dramatic reductions in mean A1c were achieved at 12 weeks (from 10.8% to 5.9%), and these benefits were maintained in both groups out to 3.5 years (Harrison et al., Diabetes Care 2012). Dr. Raskin interpreted this result as a testament to early insulin therapy’s benefits, regardless of the follow-up therapy. He did not mention ORIGIN during his talk, though in the rebuttal period he seemed to suggest that the trial was conceptually flawed.
- Dr. Raskin believes that GLP-1 agonists might preserve beta-cell function, but that to date, clinical trials have not been well designed to answer the question. He briefly reviewed preclinical evidence, and he then moved onto “the only halfway-decent study” that he could find in humans (Fehse et al., J Clin Endocrinol Metab 2005) – a crossover-design trial in which 13 people with type 2 diabetes were given intravenous infusions of either saline or exenatide, during an IV glucose tolerance test. With the saline solution, the patients had significantly depressed first- and second-phase insulin response, compared to people without diabetes. However, the exenatide infusion significantly augmented both phases of the insulin response.
Jack Leahy, MD (University of Vermont, Burlington, VT)
Presenting the con opinion, Dr. Jack Leahy argued that at this point in time, no drug or combination therapy has been proven to prevent, reverse, or stabilize the beta cell dysfunction typified in type 2 diabetes. He first offered his definition of beta cell preservation: proof of stopping the progressive decline in beta cell function, with meaningful beta cell function after drug discontinuation. Dr. Leahy then highlighted the challenges to preserving beta cells, noting that beta cell function is substantially reduced before a person becomes glucose intolerant. Thus, HCPs need a marker to identify people with compromised beta cell function before they seek treatment for hyperglycemia. Dr. Leahy emphasized that glucose toxicity impairs beta cells and discussed the potential of current anti-diabetes therapies. While ADOPT suggested that TZDs may – to a small degree – preserve beta cell function, their side effects preclude widespread use. A Chinese study (Weng et al., Lancet 2008) showed that intensive insulin therapy immediately following diagnosis improves beta cell function, but Dr. Leahy questioned whether this effect would occur in other ethnic populations. While ORIGIN showed some protective effects for Lantus, Dr. Leahy did not find the results particularly impressive. Ending with incretins and anti-inflammatory therapies, he pointed to several studies that showed that the improvement in beta cell function was either transient (stopping after drug discontinuation) or extremely small.
Dr. Leahy highlighted several challenges to preserving beta cell function. First, beta cell function is markedly impaired before glucose intolerance, suggesting that an intervention to preserve beta cell function must occur very early on in dysglycemia. Dr. Leahy cited data from Dr. DeFronzo that showed that people lose 60% of beta cell function before reaching the edge of normal glucose tolerance. Other studies show that people with prediabetes have already lost 40% of their beta cell function on average; since beta cell mass continues to decline as the disease progresses, HCPs will need therapies that work over decades. These findings highlight the need to identify people with compromised beta cell function before they exemplify the clinical symptoms of dysglycemia; however, no markers for such identification currently exist.
Dr. Leahy remarked that TZDs are the most successful therapies that have been published for preventing diabetes; however they are far from perfect. Due to TZD’s side effects, Dr. Leahy finds it unlikely that HCPs will use the drugs for prediabetes. Turning to diabetes, he cited the ADOPT study, which compared glyburide, rosiglitazone, and metformin in~4,4000 patients with recent-onset diabetes over roughly four years. While ADOPT showed thatTZDs may provide some preservation of beta cell function, the results are neither clear nor conclusive. Dr. Leahy concluded that “TZDs are not so bad, but they’re not going to be the answer” to beta cell preservation, in part due to their side effects.
The insulin pathway overlaps with pathways that regulate beta cell proliferation, differentiation, and survival, suggesting a role for insulin in preserving beta cell function. However, Dr. Leahy noted that data so far has not been especially compelling. A Chinese study found that treating type 2 patients with insulin immediately following diagnosis led to significant improvements in beta cell function during the weeks of insulin therapy and immediately thereafter. One year later, the patients who remained in diabetes remission exhibited substantial improvements in beta cell function. However, Dr. Leahy questioned the generalizability of the results, noting that the literature on intensive insulin therapy and diabetes remission focuses mostly on Asians. Thus, additional studies are required to see if the results can be achieved in other ethnic populations. Turning to ORIGIN, he noted that insulin glargine was shown to provide some protective effects against progression to diabetes. However, he commented “I don’t think I would go to bed excited” due to the relatively small group differences– 35% of people in the glargine group progressed to diabetes compared to 43% of those on standard care, an advantage he characterized as observable but not revolutionary. Dr. Leahy expressed doubt that intensive insulin therapy in prediabetes is the end solution.
Studies have investigated beta cell function following one year of exenatide or vildagliptin therapy. Both incretin therapies provided substantial but transient improvements in beta cell function – no change was observed after the drugs were discontinued. A three-year study of exenatide found that the drug provided an extremely small improvement in beta cell function that persisted after drug discontinuation; however Dr. Leahy did not appear to find the result conclusive or compelling.
Dr. Leahy ended his presentation by briefly discussing therapies for islet inflammation. He cited a study that showed that interleukin-1 receptor antagonist therapy provided a modest improvement in beta cell function over 13 weeks in type 2 patients. However, no effect was observed 39 weeks after drug discontinuation, indicating that the improvement was not durable.
Jack Leahy, MD (University of Vermont, Burlington, VT); Philip Raskin, MD (University of Texas Southwestern Medical Center, Dallas, TX)
Dr. Robert Vigersky (Walter Reed Army Medical Center, Washington, DC): Dr. Raskin, you didn’t mention ORIGIN. Dr. Leahy pointed out that the results weren’t much to write home about.
Dr. Raskin: They wasted a whole helluva lot of money, is my opinion; I don’t know why they did it this way.
Dr. Vigersky: The statistics in ORIGIN on who reverted to diabetes – that was measured at 100 days after insulin was stopped. The difference was about 35% to 45%. My guess is that if both groups were studied a year later or multiple years later, there would have been no benefit to early use of basal insulin except maybe days on other therapy.
Dr. Leahy: We could argue for hours about the best measure of insulin secretion. It is so complicated – there are so many physiologic modulators. ORIGIN was not a beta-cell function/protection study; there were just measures built in to the larger study. Studies need to be done to repeat the Weng research in non-Asians, to see if the results are applicable in people of European and Hispanic descent. The study needs to be redesigned in a reasonably applicable way. The concept that you put them on insulin and they never get diabetes again is crazy.
Dr. Raskin: We didn’t measure insulin secretion at baseline in our study, but the mean incoming A1c was nearly 11%. You don’t have an A1c of 11% if you have enough insulin. To use the HOMA model, you measure insulin and blood glucose and make a calculation – you are measuring a dynamic thing with a static measurement.
Dr. Leahy: I am not critiquing the concept. I live in the world of glucotoxicity like you do; if you improve blood glucose, it will improve beta-cell function, especially early in the course of disease. For people who say that a decrease in beta cell mass is the only problem – that’s clearly disproven by what we said. You will get beta-cell response back to a certain level if you improve glucose concentration, and you want to keep it there for a long time.
Dr. Raskin: One way to do that is to continue the treatment. No one wants to take insulin to preserve insulin function, but the truth is that it does. In type 2 diabetes, you can put patients on insulin, and add metformin – an oral drug with few side effects. TZDs are terrific insulin sensitizers, and they prevent insulin inflammation and progression to prediabetes, but I’ve stopped using them [Editor’s note – presumably because of the side-effect profile].
Dr. Leahy (speaking concurrently with Dr. Raskin’s positive comments on TZDs): I’m not going to use them, though.
Dr. Leahy: I started by saying that if someone brought us a drug – you look three years out and good beta- cell-function effects – my argument becomes this idea: People think that if they have a sustained therapy, it must be doing something magical to the beta cells. But if you take it away, the badly damaged beta cell is still there. We want to find therapies that restore beta cells to health and to keep them at that level over time.
Q: I’m surprised no one has mentioned surgery. The title of the debate says “current therapies”. Surgery has been around for 20 years, and we’ve known its effect for two decades. Studies show that you can put patients into remission for five years or longer. Studies show that you can reverse beta cell defects. Why isn’t that considered a standard therapy, and what are your thoughts?
Dr. Raskin: It is. I thought about it. The problem is that, to be honest, I don’t understand it. I don’t understand how or why. I just can’t make a judgment on it because it’s like hocus pocus to me. It works. It does. There’s no argument with that. I think there’s recidivism with that too. Even after the immediate recovery, they get the diabetes back over a certain period of time. It’s not lifelong. It would be great fix beta cells in such a way that you give it a drug and it’ll come back to normal, but I say good luck with that. But I think that what you want is a treatment that isn’t worse than the disease. For example, with a GLP-1 agonist, you can give a bit of that once a day or once a week and you get good insulin secretion, no hypoglycemia, and no weight gain.
Dr. Leahy: I agree with everything the questioner said. You guys are one of the most visible institutions publishing on this. If you tell us about the effect of weight loss surgery on the biology of type 2 diabetes, it’s pretty magical. But it’s not for every patient. It’s a complicated therapy and expensive therapy for many people. But I don’t think we have anything else that one could argue could stabilize beta cells. There’s biology that we don’t understand. We’re still trying to figure it out. So I think you’re right. Yes, I apologize – it should be on our list. But it is different than taking a pill.
Q: There’s risk. But the mortality risk is not much different than common surgeries. It has a mortality rate of 0.02%. Two in one thousand. There’s data – SOS data – showing long term that you can reduce the longer-term cardiovascular morality by 30%. I suggest we take a closer look. Some studies show a 60% five-year remission rate with no drugs and normoglycemia.
Dr. Vigersky: No one has mentioned the lifestyle arm of the DPP trial. In some ways that’s the lifestyle equivalent to bariatric surgery.
Q: One very important feature is beta-cell rest.
Dr. Leahy: You are giving me chest pain, because early in my career I used diazoxide in part because of 1960s studies by Robert Turner. Promising results were also shown by Peter Butler – I hate saying his name because we are in Southern California, and it always comes up – using somatostatin analogs. I have been a true believer in beta-cell rest. But problems with these therapies were seen even in potassium-channel openers that aren’t quite as toxic as diazoxide; the approach didn’t work out as magically as thought.
Audience Response Question (n=48): The beta cell can be preserved with currently available therapy in type 2 diabetes
- True: 29%
- False: 71%
FOXO1, INSULIN ACTION, AND METABOLIC EFFECTS
Domenico Accili, MD (Columbia University, New York, NY)
Dr. Domenico Accili gave an engaging presentation on FoXO1, a transcription factor that appears to be inactive in the absence of diabetes, active in stressed beta cells, and absent late in the course of type 2 diabetes. Dr. Accili proposed that FoXO1’s role is to maintain beta-cell homeostasis and mitochondrial function, even in the presence of glucotoxicity and high demand for insulin. However, eventually gluco- and lipotoxicity cause Fox01 proteins to be degraded. In the absence of FoX01, beta cells appear to de- differentiate into progenitor endocrine cells. (He explained that this de-differentiation – not outright beta-cell death – seems responsible for the initial loss of beta cell function in type 2 diabetes.) Dr. Accili also showed unpublished data suggesting that FoXO3a and FoXO4 play complementary roles to FoxO1. However, he emphasized that he does not think the FoXO family to be uniquely important in the progression of type 2 diabetes. Rather, he expects that many other transcription factors will be found to play similar roles in preserving beta-cell function and identity.
Inflammation, Macrophages, and ER Stress
ER STRESS, THE TERMINAL UPR, BETA-CELL SUICIDE, AND DIABETES MELLITUS
Feroz Papa, MD, PhD (University of California San Francisco, San Francisco, CA)
In his enlightening talk, Dr. Feroz Papa discussed how ER stress results in beta cell death. As the first step in the secretory pathway, the endoplasmic reticulum (ER) contains cellular machinery that folds proteins into their proper 3D structure. Dr. Papa noted that traffic through the ER is high – under healthy conditions, each beta cell makes a million molecules of insulin every minute. ER stress occurs when the demand for protein folding outweighs the ER’s folding capacity. This imbalance results in the accumulation of unfolded proteins, triggering a set of signaling pathways called the unfolded protein response (UPR) that works to restore homeostasis (“adaptive UGR”). However, under conditions of high and chronic ER stress, the UPR is unable to contain ER stress and instead triggers cell death (“terminal UPR”). The continuation of this process in the absence of cell replenishment could lead to several diseases including diabetes. Terminal UPR endpoints have been observed in degenerating pancreatic beta cells and mutations in proteins involved in terminal UPR have been associated with diabetes; these findings indicate that terminal UPR plays a role in beta cell death and reduced beta cell mass. Dr. Papa remarked that under this theory, beta cells are active participants in their own death and points to the therapeutic potential of intervening in terminal UPR. Specifically, he highlighted the importance of modulating the “switch” that decides whether UPR promotes homeostasis or cell death (apoptosis). In the last portion of his presentation, Dr. Papa reviewed studies on Ire1α, a kinase/RNase that typically dimerizes to promote adaptive UGR, but that can – under higher ER stress – oligimerize to trigger terminal UGR (via the pro-apoptotic protein TXNIP). Given its role in regulating UGR, Ire1α may represent a potential therapeutic target.
IMMUNOLOGIC REGULATION OF ADIPOSE TISSUE FUNCTION
Anthony Ferrante Jr., MD, PhD (Columbia University, New York, NY)
Seeking to clarify the immune system’s role in the metabolic syndrome, Dr. Anthony Ferrante Jr. and his colleagues have found that non-inflammatory immune effects may be more important than generally thought. He reviewed that the metabolic syndrome is commonly thought of as an inflammatory condition, but genome-wide association studies in people with type 2 diabetes have not implicated many inflammatory pathways, and anti-inflammatory therapies have generally not been very effective in treating type 2 diabetes. By contrast, Dr. Ferrante’s group has looked at white adipose tissue and found strong associations between the metabolic syndrome and genes associated with lysosome biogenesis. Evidently these additional lysosomes help to break down the excess lipids that build up in obese people’s white adipose tissue macrophages. Dr. Ferrante hypothesizes that lysosomes are probably also important in other white adipose tissue cells in obesity; he concluded that our picture of immuno-metabolic cross-talk remains incomplete.
Glucose Metabolism, Beta Cell Biology and Cancer
THE PUTATIVE ASSOCIATION BETWEEN GLARGINE AND CANCER
James Meigs, MD (Massachusetts General Hospital, Boston, MA)
Revisiting his talk from the ADA 2012 symposium on insulin and cancer risk, Dr. James Meigs concluded with confidence that insulin glargine’s clinical benefits outweigh its potential harms – a conclusion he reached from ORIGIN and from three large observational studies conducted since the glargine-cancer scare began in 2009. However, softening his more certain tone at ADA 2012, he added that the exact relationship between glargine and cancer remains unclear, in the absence of 10-to-15-year data on new insulin users. Dr. Meigs said that to clarify the issue, some researchers are thinking of linking glargine studies to cancer registries (similar to the research that suggested the cancer-protective effects of aspirin). He also emphasized the variety of reasons for which “insulin cancer risk research is extremely hard to do correctly.” On this note, he advised that when looking at any future study about a drug’s potential harm: “read the methods section before you read the results” – sage advice in a world that loves Tweet-sized headlines.
To preface his talk, Dr. Meigs said, “This disclosure is important” and noted that glargine’s manufacturer, Sanofi, funded much of the research cited in his presentation through a large grant to Dr. Meigs’ institution, Massachusetts General Hospital. (Editor’s note: We do not regularly report on presenters’ disclosures, but we also do not often hear disclosures announced with such gravity.)
Dr. Meigs reviewed “clinical study design basics,” emphasizing most of all that observational findings cannot establish causality.
Exposure to the substance of interest (glargine) should be measured accurately and should precede the outcome of interest (cancer). By this criterion, databases of new insulin users are preferable to databases of prevalent insulin users (since the exposure is not well characterized in the latter).
The outcome should also be measured accurately, and the period of substance exposure should be long enough to allow time for disease development. Dr. Meigs explained that someone who had not been to the doctor in a while, might be prescribed glargine and soon thereafter diagnosed with pre-existing cancer – a potential confound. Another methodological question is whether to define cancer based on cancer registries or cancer claim codes.
Another key issue is how to define a study’s denominator – the population of people who could be at risk. Dr. Meigs remarked that the denominator was not clearly defined in the controversial 2009 Hemkens et al. paper in Diabetologia, which analyzed a subset of patients from a German registry.
“Confounding by indication” can compromise study validity, as well. Dr. Meigs noted that glargine might be more disproportionately likely to be prescribed to people with type 2 diabetes who are especially unwell – e.g., the obese, unhealthy eaters, users of alcohol, smokers. However, these factors are all independent risk factors for cancer, and so might stack the deck against glargine.
Bias – a structural result of missing data – cannot be controlled for with statistical methods, said Dr. Meigs. The more missing data, he explained, the greater the bias – a problem for many databases, in which confounding variables such as smoking might not be reported.
Dr. Meigs summarized several presentations on insulin-cancer risk from ADA 2012, based on which he concluded that glargine’s cancer risk appears equivalent to that of alternative therapies. To the clinical question of whether insulin glargine is safe to prescribe, Dr. Meigs repeated the answer that he gave in a similar talk at ADA 2012 – a resounding “Yes!” His interpretation is that the benefits outweigh the potential harm.
He first reviewed three large observational studies on glargine and cancer risk (using data from Inovalon; Kaiser Permanente; and a series of Northern European databases). Dr. Meigs said that each of the observational studies had design limitations (e.g., an average of only a few years of follow-up in any the studies). He also noted that breast cancer rates appeared higher among new glargine users exposed to the drug for two years or more in the Kaiser database, and in some analyses of the Northern European databases. (He mentioned that the signal was not statistically significant in the Kaiser study, though this seems like a given – as we understand it, the signal would have to be huge to achieve statistical significance with such a low event rate.) All in all, however, Dr. Meigs suggested that the data assuage concerns about glargine carcinogenicity. The relative risk of cancer was 1 between glargine and NPH in both the Kaiser and Inovalon studies, and Dr. Meigs interpreted the Northern European database to show no association of glargine use with any form of cancer.
Dr. Meigs also summarized ORIGIN, Sanofi’s large, randomized clinical trial of glargine vs. standard antihyperglycemic therapy among patients with cardiovascular risk factors as well as type 2 diabetes, impaired fasting glucose, or impaired glucose tolerance (NEJM 2012). He opined that ORIGIN had “a pretty clean design,” adding, “with a few caveats, you could view it as a study of insulin treatments for cancer risk.” Roughly 12,500 patients were followed for roughly five-to-seven years. At study’s end, the groupswere essentially identical in their incidence of all cancers, breast cancer, lung cancer, and colon cancer. Dr. Meigs concluded that no “consistent risk signal” has been found linking glargine (or any other insulin) with cancer in general, or with breast, colon, or prostate cancer in particular.
For detailed coverage of ORIGIN results at ADA 2012, see our dedicated Closer Look at http://www.closeconcerns.com/knowledgebase/r/10be2669. For more on the insulin- cancer symposium featuring the three database studies – as well as Dr. Meigs’ talk during the same symposium, in which he proclaimed that ORIGIN had put “the stake in the zombie that is insulin glargine and cancer risk” – see our ADA 2012 Insulin Therapies report at http://www.closeconcerns.com/knowledgebase/r/e91ac108.
Dr. Meigs concluded by considering a nagging question: “Why are we having such a hard time being completely convinced?” He proceeded to list several reasons why definitive cancer pharmacoepidemiology research is so difficult. For starters, when the latency period is long (as for cancer), causality is hard to detect even if it is present. Also, given the low rates of cancer in the population, the exposure must be strong to detect a signal. Another hurdle is that patients are often exposed to glargine only intermittently, or only for a short time, depending on their insurance coverage or their healthcare team’s recommendations. (Speaking for clinicians, Dr. Meigs explained that “we don’t put people on medications and cling grimly to them no matter what.”) Meanwhile, competing hazards such as cardiovascular disease or other health crises might occur, further obscuring causality. Dr. Meigs also addressed the clinical reality that for people diagnosed as needing insulin therapy, the question is “not whether but which” – and it would be unfeasible to conduct a “glargine-vs.-nothing” trial for 10 years in people without diabetes.
GLUCOSE METABOLISM, CHREBP, AND PROLIFERATION IN THE BETA CELL
Donald Scott, PhD (Mount Sinai School of Medicine, New York, NY)
Dr. Donald Scott’s engaging presentation detailed how the carbohydrate response element binding protein (ChREBP) drives beta cell proliferation in response to glucose. He opened by explaining the finding that high blood glucose levels promote beta cell replication by increases the amount of work each beta cell must perform to maintain normoglycemia. ChREBP is a transcription factor that resides in the cell cytoplasm during levels of low glucose. High levels of glucose activate ChREBP (via an unknown metabolite), allowing it to enter the nucleus, interact with its binding partner MLx and co- activators, and alter the transcription of glucose-responsive genes. Dr. Scott emphasized that this process is highly dependent on glucose metabolism. Studies show that ChREBP is expressed in beta cells and is required for glucose-stimulated beta cell proliferation. Furthermore, overexpression of ChREBP amplifies the glucose response in islets. Further investigation into the mechanism of ChREBP revealed that ChREBP increases the levels of the protein Myc, which in turn upregulates cell cycle regulators (i.e., cyclins D2, A, and E) to drive beta cell proliferation. In ending his presentation, Dr. Scott highlighted that the glucose/ChREBP/Myc/cyclin pathway may be a new therapeutic target for diabetes.
PTEN AT THE INTERSECTION OF DIABETES AND CANCER
Bangyan Stiles, PhD (University of Southern California, Los Angeles, CA)
In a thoughtful and well-structured presentation, Dr. Bangyan Stiles discussed the role of phosphatase with tensin homology (PTEN) in liver tumorigenesis. PTEN is a negative regulator of AKT 1,2,3 and decreasing PTEN results in increased levels of AKT 1,2,3, as well as elevated level of PI3K, a positive AKT 1,2,3, regulator. The signaling of PTEN and/or PI3K on AKT 1,2,3 regulates several pathways including those involved in cell survival, cell growth, protein translation, transcription, cell metabolism, senescence. Focusing on cell metabolism, Dr. Stiles explained that deleting PTEN expression in the liver of mice resulted in fatty liver, fibrosis, and tumor development. She then reviewed a series of experiments that led to a two-hit hypothesis of how Pten deletion results in tumorigenesis: first, the loss of Pten triggers a “proliferation signal” in progenitor or progenitor-like cells that transforms them into tumor-initiating cells. Such transformation is insufficient by itself for tumor growth, as a “niche activation signal” is required for these cells to expand. This signal is offered by Pten deletion in hepatocytes: Pten deletion activates AKT and results in oxidative stress (via CREB and ERRα) and lipid accumulation (via AKT2 and FOXO1), which cause hepatocyte death and produces a signal that allows the already-transformed cells to develop into tumors. Dr. Stiles ended her presentation by discussing PTEN’s effect on progenitor cells, noting that Pten deletion in islets may actually be beneficial by increasing beta cell mass, likely via alterations in p16 and cyclins.
RLIP76: CONNECTING METABOLIC SYNDROME, OBESITY & CANCER
Sanjay Awasthi, MD (City of Hope, Duarte, CA)
Dr. Sanjay Awasthi reviewed preclinical research demonstrating that mice lacking the mercapturic acid pathway transporter protein, RLIP76, are resistant to chemical carcinogenesis, inflammation, and the metabolic syndrome. Notably, these mice are so sensitive to insulin they require double the level of glucose, than control mice, to maintain their blood glucose. Dr. Awasthi encouraged researchers to consider targeted depletion or inhibition of RLIP76 as a novel option for the treatment or prevention of both obesity and cancer.
Sanjay Awasthi, MD (City of Hope, Duarte, CA); James Meigs, MD (Massachusetts General Hospital, Boston, MA); Donald Schott, PhD (University of Pittsburgh, Pittsburgh, PA); Bangyan Stiles, PhD (University of Southern California, Los Angeles, CA)
Q: Everybody comes from countries with free speech yet you are not allowed to cry fire in a movie theatre. How do we allow scientific reporting to occur without crying theater in a crowded movie theater? A recent example is a study finding an association of GLP-1 agonists with pancreatic cancer. What is the responsible way to proceed in these situations?
Dr. James Meigs: There are two components. The first is patient protection; we want to make sure that we are doing no harm. We need to take safety signals seriously. The second is the responsibility of journal editors. Different people get to be journal editors at different levels for different reasons. I think that the effort of scientific reporting is one of honor and good practice; all we can do is keep looking out for that. The consumers of scientific literature need to know what kind of study designs we can believe in or not. We need to trust the peer review process. Rumor has it that the reviewers of the Diabetologia papers did not want them to be published but they were. It is helpful for scientists to know the methodological approaches in the area you are reviewing.
Q: Insulin is a tropic factor. Should the discussion be that maybe we should add something to insulin treatment, such as metformin, which has been shown to reduce cancer risk?
Dr. Meigs: Metformin has been associated with low risk of cancer. I am not sure we can say that it is preventive. Second, keeping in mind the limitations of jumping from studies to clinical practice, I think we want to know which drug regimen will provide the best outcomes. Along these lines, there is a comparative-effectiveness study called GRADE that is starting up.
Q: I was interested in the mitochondrial response you saw, did you characterize how well coupled the mitochondria were?
Dr. Stiles: We measured the oxygen consumption and also the lactate production. What we see is the coupling is still there with PTEN. PTEN seemed to regulate both of them independently. They still respond when you put in the mitochondrial poisons?
Q: Did you see any activation of AMPK?
Dr. Stiles: AMPK is very confusing. We went down that way a few years ago and then had to stop because it was very confusing.
Dr. Robert Vigersky (Walter Reed Army Medical Center, Washington, DC): You recall, that Hemkens paper was actually held for a year until Diabetologia commissioned the three other papers that were all published together, to give some balance. That was a very savvy move, and I think it lent some credence to the issue that this was not a signal.
One issue that you didn’t mention is, what’s the relative benefit vs. risk. That’s something I don’t see in any of these papers. These are looking at relative risk, but not relative benefit.
Dr. Meigs: I think there have been about 50 opinion pieces written about the Diabetologia thing was handled. It was highly unusual for a journal like Diabetologia to commission papers. The problem is that even many good papers will not outweigh one bad paper. The popular paper will pick up these things and go crazy. I think that the prominent journals need to be very careful about what they pick up and not be motivated by political or other agendas.
People with type 2 diabetes die from cardiovascular death. They die earlier. Their risk rates are higher. People with ty2d are still twice as likely to die from cardiovascular disease. That was what ORIGIN was about; does glargine have an advantage? And it found that there was no advantage over usual care. So I think that it is more complicated than that.
Q: I might caution the use of ChREBP as drug target, mainly because in type 2 diabetes it seems already to be fully activated and to mediate some of the gluco- and lipid toxicity.
A: You’re absolutely right, and I’m very aware of the work you did with overexpression that killed beta cells. My guess is that it’s probably the beta form; in our studies we overexpressed the alpha form. That said, type 2 diabetes is a hyperglycemic state, so absolutely we have to be cautious of this.
RODENT MODELS OF EPIGENETIC REGULATION OF INSULIN RESISTANCE AND DIABETES
Rebecca Simmons, MD (University of Pennsylvania Center for Research Reproduction & Women’s Health, Philadelphia, PA)
Dr. Rebecca Simmons discussed several studies investigating the epigenetic regulation of diabetes- related proteins. She opened by discussing the established association between diabetes and intrauterine growth retardation (IUGR; in which a baby is born at low weight). Turning to non-genetic animal models, she described an IUGR mouse model (bilateral uterine artery ligation) in which the mice are born with low birth weight and develop fasting hyperglycemia, impaired beta cell proliferation, and diabetes-like phenotypes. Further investigation into gene expression indicated that Pdx1, whose expression is reduced in IUGR, plays an important role in the beta cell changes observed in IUGR mice. Dr. Simmons then detailed a series of studies examining the epigenetic regulation of Pdx1, which indicated a role for histone modification. Specifically, altered histone modification in islet cells appeared to be caused by the complete loss of binding between USF1 (a protein required for normal histone modification patterns) and the Pdx1 gene region. Dr. Simmons ended her presentation by discussing type 2 diabetes as an altered epigenetic landscape.
THE USE OF AN AUTOMATED GLUCOSE CONTROL SYSTEM FOR OVERNIGHT GLUCOSE CONTROL IN ADOLESCENTS WITH TYPE 1 DIABETES
Timothy Jones, MD (Princess Margaret Hospital, Perth, Australia)
Dr. Timothy Jones gave a very high-level talk on closed-loop insulin delivery, highlighting the work at Princess Margaret Hospital with Medtronic’s Portable Glucose Control System (PGCS). He reviewed the team’s overnight study (n=8) published in Diabetes Care (Ly et al., 2011) and the treat-to-range data just presented at ATTD 2013 (see link below) – both studies demonstrated feasibility and excellent overnight control. However, he reiterated that the daytime period is still challenging (e.g., in the most recent study, treat-to-range control was no better than open loop therapy). Dr. Jones looks forward to future studies of the PGCS treat-to-range system, which will include moderate intensity exercise and missed meal boluses. Dr. Jones also discussed the major current goal of researchers – moving closed- loop control into the outpatient setting – and characterized the DREAM-3 camp study’s recent publication in the NEJM as a “milestone” (Phillip et al., 2013). In his view, closed-loop control represents the first advance in diabetes technology that actually improves control while reducing the burden of care – we thought this was a good point, though we believe pumps and CGMs still have room to improve in this domain. Like Dr. William Tamborlane’s “Pitfalls in the Development of the Artificial Pancreas” presentation at ADA Post Grad 2013, Dr. Jones believes the major obstacle is safety. He concluded with a review of the Medtronic Veo, which has “little or no risk and potentially great benefit.” Results from the Australian Low Glucose Suspend study, testing the effect of the Veo insulin pump on severe hypoglycemia, will be presented at ADA 2013 in Chicago.
- For more details on the treat-to-range study of Medtronic’s PGCS, please see pages 50- 51 of our ATTD 2013 report at: http://www.closeconcerns.com/knowledgebase/r/16e1264b.
THE PERITONEAL CAVITY AS A SITE FOR GLUCOSE SENSING: A FEASIBILITY STUDY
Daniel Burnett, MD (TheraNova, San Francisco, CA)
Dr. Daniel Burnett discussed TheraNova’s feasibility study testing CGM sensing in the peritoneal cavity. Modified Dexcom Seven sensors were tested in eight nondiabetic pigs over 15 intravenous glucose tolerance tests. The study was concerned with lag time (accuracy figures were not reported), and indeed, intraperitoneal latency was shorter than subcutaneous latency with respect to time to half maximal values (p <0.001). Dr. Burnett believes the peritoneal space demonstrates “excellent potential,” which will be examined more fully in an upcoming ambulatory study in human patients with type 1 diabetes. The study will compare subcutaneous sensors to intraperitoneal sensors over 14 days. Both accuracy (MARD vs. YSI) and lag time will be measured at in-clinic glucose challenges. The trial’s protocol is currently being formed and TheraNova hopes to begin the trial in the next month or two. Notably, Drs. Howard Zisser and Eric Renard will be involved. We think TheraNova’s data is very early stage at this point, though we certainly look forward to seeing results (especially on accuracy) from the upcoming clinical study. In our view, CGM lag time is definitely not the biggest barrier in closed-loop delivery, though it’s good to see companies working to improve it.
SYSTEM AND CONTROL ENGINEERING FOR TYPE 1 DIABETES
Frank Doyle III, PhD (University of California Santa Barbara, Santa Barbara, CA)
Summarizing his team’s decade-plus of work on closed-loop glucose control, Dr. Frank Doyle III reviewed technical challenges and offered practical insights (e.g., FDA was wary of a control algorithm that was submitted as a series of abstract equations, but the agency was much more comfortable when the algorithm was presented as a look-up table that showed how the system would dose insulin based on any glycemic measurements). Dr. Doyle also presented clinical data – including encouraging results from pilot studies of closed-loop systems that incorporate non-traditional forms of insulin delivery (i.e., Roche’s DiaPort, MannKind’s Afrezza). For more on these results, unveiled in Paris less than two weeks earlier, see our ATTD 2013 coverage at https://closeconcerns.box.com/s/v66m2tpgges6k8hzsvk2.
Questions and Answers
Q: How are you modeling exercise?
A: We haven’t yet built patient models for that; we are treating exercise as an un-modeled disturbance. The algorithm seems to handle it without needing a specific patient model. The patient model does include specific information on insulin requirements, however, such as insulin-to-carbohydrate ratio.
Q: Have you studied the APS in patients with well-controlled type 1 diabetes? The before- and-after slides showed very large glycemic swings in the “before” studies.
Dr. Zisser: Patients’ incoming control is not really the issue. You see the large excursions because we have the system respond in real time, without any meal announcement or mealtime bolusing, for both study conditions. You’ll have poor control [with the subcutaneous pump alone] and better control [with the introduction of Afrezza or the use of the DiaPort]. As we optimize the controller, all of those excursions should come down.
INTENSIVE INSULIN THERAPY USING AN ARTIFICIAL PANCREAS WITH CLOSED-LOOP SYSTEM
Kazuhiro Hanazaki, MD (Kochi Medical School, Kochi, Japan)
Dr. Kazuhiro Hanazaki discussed inpatient, closed-loop intensive insulin therapy with the Nikkiso Company’s STG-55, a bedside artificial pancreas device that measures peripheral blood glucose levels and automatically delivers intravenous insulin and intravenous glucose. In prospective, randomized trials, intensive insulin therapy with the STG-55 significantly reduced surgical site infections and shortened length of stay among patients undergoing hepatectomy and pancreatetectomy; moreover, a target range of 80-110 mg/dl was found to be significantly better than 140-160 mg/dl. Like Dr. Anthony Furnary’s work in cardiothoracic surgery, this research demonstrates that tight glucose control in the hospital can be performed with minimal hypoglycemia and dramatic clinical benefits after surgery. The STG-55 is commercially available in Japan but has neither FDA-approval nor CE-mark.
The STG-55 (like its precursor, the STG-22) is a bedside device for closed-loop glucose control in the hospital. It continuously monitors blood glucose levels by sampling <2 ml of venous blood every hour, and it intravenously delivers insulin and glucose to maintain tight glycemic control. In multiple trials, the STG-22 has been reported to maintain glucose levels at a low mean (~100 mg/dl), with no hypoglycemia and with low variability (Yatabe et al., Crit Care Med 2011). In randomized controlled trials, closed-loop control compared favorably to sliding scale insulin dosage. In patients receiving pancreatectomy (Okabayashi et al., Arch Surg 2009) or hepatectomy (Hanazaki et al., Ann Surg 2009), the incidence of surgical site infections was significantly reduced in the postoperative period, and postoperative length of stay was significantly shorter. Because of these benefits on outcomes, the researchers found that using the STG can actually save costs compared to sliding scale insulin (Meyfroidt et al Crit Care Med 2010).
- An STG artificial pancreas was used in a randomized clinical trial to compare two target glycemic ranges (80-110 mg/dl vs. 140-160 mg/dl) in patients admitted for hepatectomy and pancreatectomy. Notably, the lower target led to significantly fewer surgical site injections and significantly shorter post-operative stay (data under submission).
Questions and Answers
Q: Is there a difference in glycemic control in the entire post-operative period, not just the first day?
Howard Zisser, MD (Sansum Diabetes Research Institute, Santa Barbara, CA); Frank Doyle III, PhD (University of California Santa Barbara, Santa Barbara, CA); Timothy Jones, MD (Princess Margaret Hospital, Perth, Australia); Kazuhiro Hanazaki, MD (Kochi Medical School, Kochi, Japan); Daniel Burnett, MD (TheraNova, San Francisco, CA)
Q: What are your thoughts on implantable sensors?
Dr. Zisser: We tested a long-term sensor that sat in the superior vena cava. Ten or 11 patients had the sensor for 12-18 months. The sensors had a significant time lag – it was the result of special coating that allowed the sensors to stay in the blood stream for that long. Twenty to 25 minutes of lag is David Gough’s work. I’m not familiar that they did peritoneal sensing.
Q: We did a study with Medtronic and used feedback control, but it did not work as well as PID. It sounds like feedback control is working better now.
Dr. Zisser: PID is what Gary was working with, but they figured out insulin action and added that on top of the controller. Frankly, it was a hybrid controller that was pushing towards MPC, but not strictly. In most of these studies, overnight control will work with almost any controller. It’s really meal excursions that change the equilibrium of the system.
Q: I’m wondering about peritoneal sensing. With your experience in other peritoneal devices, what do you think is the long-term feasibility and safety of an indwelling peritoneal device? Do you envision people using it for years or changing it on a regular basis?
Dr. Burnett: That’s one of the big questions the granting agency still has – what is the frequency of replacement that would be tolerated. It will require at least a procedure. With other peritoneal devices, we have several patients that are out two to three years with catheters in place. It’s all about the placement and prep of the cavity, as well as the design of the catheter itself. A rigid catheter is a non-starter. It will perforate the bowel or bladder.
Q: In our experiments, the exercise problem is really related to insulin depot formation. I think the peritoneal approach could get around that.
Dr. Zisser: For exercise, it is an issue depending on when the exercise comes. We’re using accelerometry models to see when patients are active. We also have safety systems built in, though we don’t currently use glucagon. We just did 10 patients in France with the DiaPort, and the advantage is that you can turn insulin delivery on and off quickly. There’s no subcutaneous depot.
Audience Response Question #1: What real world factors do you think will pose the biggest challenge in outpatient trials of the AP?
Large unannounced meals 31%.
Unannounced change in insulin sensitivity: 25%Hardware: 23%
What is the most promising approach to improving insulin PK for the AP?
Faster subcutaneous insulin (new analogs): 27%
Intraperitoneal delivery: 38%
Alternative routes of administration: 11%
Insulin delivery by any of these approaches is too risky: 9%
Current insulins are fast enough: 15%
Diabetes and the Gut Microbiome
DIABETIC KIDNEY DISEASE: WHAT’S NEW AFTER ACE/ARBS?
Mark Cooper, MD, PhD (Baker IDI Heart and Diabetes Institute, Melbourne, Australia)
Dr. Mark Cooper’s presentation on diabetic kidney disease focused mostly on the promise of Nox inhibition using compounds developed by GenKyoTex, though we found his discussion of Reata’s bardoxolone methyl most interesting. He called bardoxolone’s development “a very important lesson,” since Reata did not do the basic science research that would have revealed the safety signals uncovered in the phase 3 BEACON study. We were fairly shocked to hear this, and hope to hear more details emerge on potential for the drug or other treatments addressing CKD.
The development of bardoxolone methyl is “a very important lesson” in the importance of basic science research. Originally, bardoxolone was being developed as a prostate cancer drug. While it failed for that indication, an analysis of the safety data revealed nearly all patients halved their serum creatinine. The researchers then did a “quick” follow-up study in patients with type 2 diabetes and renal impairment. The rapid increase in eGFR, Dr. Cooper said, made everyone think bardoxolone might ultimately be renal protective. According to Dr. Cooper, Reata did not collect any animal data and there was no clear mechanism of action. He also pointed out the dramatic weight loss in those using the drug, which occurred in a relatively short period of time (“any drug that reduces body weight by 10 kg – you wonder if it's a safe drug”). In his view, bardoxolone’s development is a lesson in what happens when you “rush through something” without having done the basic research: you don’t really know where you’re going.
- A recent preclinical study testing use of bardoxolone analogs in rats demonstrated worsened diabetic nephropathy with additional adverse effects. The paper (Zoja et al., Am J Physiol Renal Physiol 2012) was published online on November 7, 2012, just after Reata’s press release on October 18, 2012. (Editor’s note– we did not learn about this paper until today.) Kidneys from three rats receiving a bardoxolone analog showed the presence of a granulomatous and inflammatory process reminiscent of a pseudotumor. The paper concludes, “Altogether these data raise serious concerns on the use of bardoxolone analogs in type 2 diabetic nephropathy.”
The implications of the research are not fully clear. As we understood from Dr. Cooper’s comments, had researchers done this preclinical basic science research earlier, they may not have pursued the drug further. However, Dr. Cooper said he does believe bardoxolone may still have promise. He explained that Reata used a different formulation of the drug in the phase 3 study, and bardoxolone’s effects might be dose-dependent. In some ways, this is counter to “…they may not have pursued the drug…” and we would like to know more on this front.
- Dr. Cooper extensively reviewed Nox inhibition, explaining that it could be a great target for diabetic complications. Researchers have actively been testing Nox inhibitors developed by a company named GenKyoTex (e.g., GKT 136901 and GKT 137831). In three completely different mouse models of diabetic nephropathy, the GKT compounds effectively reduced nephropathy. Interestingly, Nox inhibition also attenuates atherosclerosis, meaning it could be a target for more than just nephropathy. Dr. Cooper believes that combination therapy with a Nox inhibitor and an ACE inhibitor could be quite promising.
GenKyoTex presented phase 1 data in 72 healthy adults on GKT137831 at Kidney Week 2012. GKT137831 was found to be safe and well tolerated when administered orally, both at single doses of up to 1800 mg OD, and at multiple doses of up to 900 mg OD for 10 days. According to its website, the company is now planning the initiation of phase 2 clinical studies, including the evaluation of oral GKT137831 in patients with diabetic nephropathy.
Questions and Answers
Q: Do ACEs and ARBs block Nox’s?
A: That’s the next question to ask. ACE inhibitors lower blood pressure, and Nox compounds have no effect on blood pressure. The real experiment is using ACE inhibitors and Nox inhibitors together to see if they have an additive, synergistic effect.
SYSTEMS BIOLOGY OF DIABETIC KIDNEY DISEASE: LESSONS FROM ANIMAL MODELS AND HUMAN DISEASE
Matthias Kretzler, MD (University of Michigan, Ann Arbor, MI)
Presenting his group’s work on diabetic nephropathy as a case study, Dr. Matthias Kretzler described a research approach that integrates functional genomics, model systems, and clinical studies. He and his colleagues have mined large datasets of patients with chronic kidney disease, in an effort to map interactions between molecular pathways and clinical disease progression. They have recently begun to translate these association studies into mouse research and even clinical trials – including an ongoing, 250-patient phase 2 study of Lilly’s oral JAK1/JAK2 inhibitor baricitinib.
- Dr. Kretzler laid out an integrated research approach that he hopes will improve understanding of disease mechanisms, foster the development of new mechanism- based therapies, and – ultimately – inform clinical decision-making (e.g., predicting which patients’ disease will progress, selecting particular therapies based on patients’ disease process rather than their diagnosis alone). This strategy involves pooling a variety of data from blood and urine samples, clinical biopsies, and gene expression assays.
Based on Dr. Kretzler’s research implicating the JAK-STAT pathway in kidney disease, Lilly has begun clinical trials of its oral JAK1/JAK2 inhibitor baricitinib in diabetic kidney disease. Baricitinib is being studied in focus of a 250-patient phase 2 trial that began in August 2012 and is slated to complete in January 2014. The primary outcome is 24-week change in urinary albumin/creatinine ratio (ClinicalTrials.gov identifier NCT01683409). The drug was first developed for rheumatoid arthritis and psoriasis. However, Dr. Kretzler said that Lilly found his group’s preclinical data compelling enough to initiate clinical research in kidney disease just 14 months after target identification – an encouraging sign that basic systems biology research can translate quickly to patients.
- Dr. Kretzler briefly mentioned Nephromine 4.0, his group’s systems biology search engine for the renal research community. As its name suggests, the search engine is a way to “mine” large datasets and uncover connections between molecular biology and clinical outcomes.
STEM CELL APPROACHES FOR VASCULAR REPAIR IN DIABETIC RETINOPATHY
Maria Grant, MD (University of Florida, Gainesville, FL)
Unfortunately, people with diabetes’ progenitor cells, such as CD34+ cells, tend to be less able to help injured tissues repair. Thus, the reparative ability of diabetic CD34+ cells must be corrected before these cells can substantially aid with the healing process. Dr. Maria Grant detailed how intermittent fasting (a pattern of eating that alternates between periods of fasting and non-fasting) could be a non- pharmacological strategy for correcting progenitor cell dysfunction and thereby aid with the repair of diabetic retinopathy. One possible pharmacological treatment, Dr. Grant highlighted was activating ACE2. People with type 2 diabetes who have normal ACE2 activation tend to not have complications, largely regardless of their glycemic control, whereas people with low ACE2 activation often develop complications even if they have well-controlled glucose levels. She hypothesized that this phenomena results from ACE2 activation maintaining progenitor cells’ reparative potential. During the subsequent panel discussion, Dr. Grant stated that there is an ACE2 activator available for cattle but that it is intolerable and unsafe for humans. Her lab is working to develop safer ACE2 activators. Finally, Dr. Grant described how mesenchymal stem cells (MSCs) and adult stem cells (ASCs) may represent novel cell therapies for diabetic retinopathy. These cell types are particularly attractive for the treatment of diabetic retinopathy because they are not immunogenic. This means that MSCs and ASCs can be derived from one source for many different people, rather than a person having to receive their own cells.
People with diabetes’ progenitor cells have reduced reparative potential. Healthy, but not diabetic, CD34+ cells repair vasodegeneration by integrating into the damaged vessels of a diabetic rat’s retina and differentiating into endothelial cells. Additionally, diabetic CD34+ cells exhibit altered paracrine (hormonal signaling between neighboring cells) function. Diabetic CD34+ cells secrete abnormally low levels of reparative and growth factors, such as hepatocyte growth factor and they produce excessively high levels of pro-inflammatory factors, hindering tissue repair.
Additionally, there are demographic variations in people’s progenitor cell reparative function. Younger people tend to have better reparative cells than older people and white Caucasians often have better reparative function than other races. Statins have been found to improve reparative potential and sleeping for eight hours a night appears to dramatically improve the function of reparative cells.
Dr. Grant detailed how intermittent fasting could be a non-pharmacological approach for treating diabetic retinopathy. In humans, endothelial progenitor cells’ (EPCs) secretion follows a circadian pattern, with the peak of EPC release occurring during rest. However, people with type 2 diabetes have a lower peak and an altered circadian pattern. Peak of release of EPC during rest – same with humans. Dr. Grant stated that this dysregulation is believed to be due to bone marrow neuropathy. A six-month mouse study of intermittent fasting found that while intermittent fasting did not affect A1c levels, it was associated with increased diabetic mouse survival rate. More specifically, intermittent fasting was tied to improved migration and proliferation of EPCs and a correction of gene expression in diabetic mouse bone marrow cells. Thus, she concluded that intermittent fasting could be a non-pharmacological therapy for diabetic retinopathy.
- Dr. Grant began her presentation explaining that progenitor cells help repair injured tissues predominately by providing paracrine support; very few progenitor cells actually differentiate into endothelial cells and integrate into the tissue.
ADVANCED GLYCATION: AGE-RAGE AND DIABETIC VASCULAR COMPLICATIONS
Ann Marie Schmidt, MD (NYU Langone Medical Center, New York, NY)
Dr. Ann Marie Schmidt discussed RAGE, the receptor for advanced glycation end products (AGE). Hyperglycemia increases the production of AGEs, and research shows that diabetes patients exhibit elevated levels of RAGE. Binding of AGE to RAGE leads to tissue damage via several mechanisms, including the increased production of reactive oxygen species and inflammatory cytokines. Dr. Schmidt first mentioned that RAGE expression in T cells may contribute to type 1 diabetes, and then focused on the role of RAGE in diabetes-related vascular complications. In diabetic mice with accelerated atherosclerosis, blocking RAGE (using a drug or genetic deletion) significantly attenuated the development of vascular plaques and atherosclerosis. The data indicate that in diabetes, RAGE may facilitate the acceleration of atherosclerosis through a unique mechanism, perhaps independent of glucose or lipids. Dr. Schmidt next highlighted that RAGE may contribute to the pathology of type 2 diabetes – in mice fed a high-fat diet, RAGE deficiency prevented hyperglycemia, increased insulin tolerance, and appeared to increase energy expenditure (without affecting food intake). Investigation into the underlying mechanisms showed that RAGE deficient mice exhibit higher levels of macrophage 2 (M2; believed to be protective against insulin resistance) but no change in levels of macrophage 1 (M1; which may promote insulin resistance); this finding suggests that the balance of M1 and M2 could be important in type 2 diabetes. Dr. Schmidt ended her talk by reviewing studies on mDia1, which interacts with the cytoplasmic domain of RAGE to mediate RAGE’s downstream effects.
Rana Natarajan, PhD (City of Hope, Duarte, CA); Ann Marie Schmidt, MD (Columbia University, New York, NY); Maria Grant, MD (University of Florida, Gainesville, FL); Matthias Kretzler, MD (University of Michigan, Ann Arbor, MI)
Q: Great talk Dr. Kretzler. Your presentation obviously explains the complexity of the disease very well. Do you have any incite into if proteomics or metabolics is a better predictor of disease?
Dr. Matthias Kretzler: I think that it is a key question and not straight forward to answer. I was very careful when discussing mechanisms, you have to be very careful in what context you are describing mechanism activation. The transcript will tell you where it is tending to go, it will not tell you what it actually does. Determining that, I think, will be informative. We are taking these transcriptional signals and seeing which proteins are discordantly regulated. Sometimes we see no association sometimes we see a correlation. If we see a correlation, I think, that we are in the position to use that signal very effectively. With proteomics we are slowly getting there. Proteomics, particularly in bodily fluids will pick up a lot of noise. What cancer has taught us is that starting with a more targeted approach using the tissue might be a more effective strategy. I think we will see more exciting results coming from metabolics. To see how we can use that as an independent domain, we took transcriptional factors and mapped the transcriptional changes regulating metabolics, to inform our colleagues to target compounds to a few hundred metabolites. Again that is a strategy that at least gives some first insight.
Q: It was great to see that systems biology data led to doing a trial of a JAK2 inhibitor. Has there been research on using this drug, or another drug, only in patients with a particular profile that suggests they would be likely to benefit?
Dr. Kretzler: That is a long-term goal of this systems biology approach. We are building these cohorts up now. We know the transcription signatures, genome sequences, and more for hundreds of patients. With the JAK/STAT pathway, it’s particularly nice – this pathway is involved in many metabolic processes that can be assessed in urine and plasma tests. We hope that this trial will be used to assess results in particular patient cohorts, in addition to the traditional analysis of the group as a whole.
Q: This is really exciting, is there any room to look at ACE2 activators and what would the mechanism be?
Dr. Maria Grant: There is any antihelminitic that activates ACE 2. Unfortunately, it can only be used in cattle; when it is used in patients it causes a lot of side effects. We are trying to identify ACE2 activators for people. Unfortunately, many people with diabetes have low ACE2 levels. We all know that serum levels and biomarkers are not always the best indicator of physiology but it at least gives you some idea. We are making some associations and in a study the NIH funded just last year we are longitudinally following patients and controls with different degrees of retinopathy for five years. We are measuring their CD34+ cells to see what is happening with those cells. It appears that if a patient can maintain their ACE2 levels they can maintain their reparative capacity and avoid complications. Some patients continue to express the ligand and the receptor; they are maintaining the reparative capacity despite having a disease that last decades. This is probably thanks to their genetics or maybe lifestyle. However, for people who do not have good genetics of lifestyle I think this could be a good treatment option. The stem cells go everywhere. If you do not treat the small vessel disease you do not treat the ischemia. That is the exciting thing about cell therapy, you can get into these smaller areas.
Q: Could you further explain the research that you mentioned on intermittent fasting?
Dr. Grant: The significance is that without changing the animals’ weight or their A1c, we were able to correct this defect in clock-gene expression. This happens not only in the progenitor cells, though I focused on these in my talk. We see the same phenomenon in the liver – we can correct the diabetes- induced defect in clock genes. By doing intermittent fasting, you can restore natural rhythms. By simply restoring the intrinsic clock function in the liver and in these other target organs, these animals are doing better and living longer, even though we are not targeting their glucose levels. Maybe the abnormalities in the clock genes occur very early in disease, even before the development of complications. It’s a non- pharmacological approach that prevents neuropathy to bone marrow. In all the models we’ve studied, we’ve shown that preventing bone-marrow neuropathy can prevent retinopathy and nephropathy down the road. You still have dysfunction, but if you have adequate repair then the animals survive better. I would expect this would contribute to fewer complications, as well.
Q: As a clinician I am always surprised that some patients have good ischemia control and have complications and others have poor ischemia control and have no complications.
Dr. Grant: The study in which we identified that the ACE2 levels were high was in patients that should have had complications – individuals that had terrible control, an A1c of 13 or 11. However, their vasculature was pristine. We age matched and sex matched them to controls. There are some very critical systems like the angiotensin system and the TGF-beta system. If the reparative cells do not succumb to the typical response – drop ACE2 – then they do not develop complications. If your reparative function is so good then you might not develop complications. We are hoping to study that in more depth in the longitudinal study.
Q: Have you looked at prediabetic individuals like obese people to see if they have CD34+ dysfunction?
Dr. Grant: This phenomenon is only in the type 2 patients. In a type 2 patient that is obese, or a person with prediabetes, if they maintain a normal TGF-beta levels then we are going to predict that they will not develop complications.
Dr. Kretzler: It is fascinating to see this tight interplay between genetics and control. They only explain a small fraction of the risk but they are starting to emerge. Indicating that we are seeing some tip of the iceberg. What we have learned in the renal field is that some people have a strong preventive risk factor but that it only manifests if you have an environmental risk factor as well. For example one risk factor present in African Americans increases your risk by about 90. That risk factor only gets operational if the person has increased BMI. We have to think about how we define our phenotypes; what do we want to know. Others do just fine with poor glycemic control while other progress rapidly. So I think that I stressed early on in my presentation good phenotypes and careful characterizations of our patients are critical for our patients to be successful. Need to have an open dialogue over the next couple of days on how to design our studies to cover these elements.
DYSFUNCTIONAL HEMATOPOIETIC CELLS MEDIATE CHRONIC DIABETES COMPLICATIONS
Lawrence Chan, MD (Baylor College of Medicine, Houston, TX)
Citing primarily preclinical research, Dr. Lawrence Chan shared an unconventional hypothesis for how diabetic neuropathy arises. The “dogma,” as he reviewed it, is that hyperglycemia causes dysregulated metabolism in mature nerve cells, leading to neuropathy. He suggested that an equally important role might be played by hyperglycemia-induced dysregulation in bone-marrow-derived cells. Experiments in rodents and in vitro suggest that in the presence of hyperglycemia, a small percentage of bone- marrow-derived cells start to express proinsulin and fuse with nerve cells. These fusion cells promote neuropathy when they are introduced into otherwise-healthy mice; in mouse models of diabetes, neuropathy abates when the fusion cells are selectively destroyed. He did not show any data on the “multi-million-dollar question” of whether similar mechanisms are at work in humans, but one of his post-docs has done nerve-biopsy studies supporting this hypothesis. Further, Dr. Chan noted that other groups have also proposed that proinsulin-producing cells can promote complications in the nerves and other organs.
Questions and Answers
Q: Could you comment on the specific population of BMDCs that you believer are fusing with the neurons? Also, are there certain dorsal root ganglion cells of which you see more fusing?
A: We have done limited analysis of the cells themselves, because the proinsulin expression is so low. It’s difficult to isolate an impurity. GFP-staining alone doesn’t do it, though we have other models that might work. We don’t really know whether they have particular types of preference for the DRG neurons. We didn’t see any suggestion in terms of size of the cell, for instance. We have some peptides that target special neurons; we haven’t done this work yet though. In general these are not stem cells.
Q: Do you see the DRG cells express more CCR2 or MCP-1? That is a typical profile in hyperglycemia.
A: No, we have not.
BEYOND GENETICS: THE EPIGENETIC CODE IN CHRONIC KIDNEY DISEASE
Dominic Raj, MD (George Washington University, Washington, DC)
Dr. Dominic Raj suggested that epigenetics may explain why chronic kidney disease is heritable but does not associate strongly with genes, per se. He noted that epigenetic changes seem to mediate the transition from epithelial cells to mesenchymal-like cells – e.g., the pattern of fibrosis that characterizes chronic kidney disease. Also, epigenetic modifications can be induced by hyperglycemia. This suggests that epigenetics may underlie the phenomenon of “metabolic memory” in DCCT, whereby a period of intensive glucose control leads to better microvascular outcomes many years later. Dr. Raj hopes that because epigenetic changes are reversible, they might offer a good target for disease-modifying therapies. Indeed, he showed preclinical data on an inhibitor of microRNA-192 that significantly reduces renal fibrosis (Putta et al., J Am Soc Nephrol 2012).
- Dr. Raj reviewed that field of epigenetics, which involves “heritable change in the pattern of gene expression mediated by mechanisms other than the primary nucleotide sequence.” He indicated that epigenetic modifications involve transcription, the transition from DNA to RNA (which in turn is translated to proteins). These epigenetic changes fall into three main categories. The first is modification of the histones that determine how DNA is packed in the cell (i.e., which genes are exposed for transcription); these histone modifications include acetylation, phosphorylation, and methylation. The second category is DNA methylation, which also affects how transcription-related proteins interact with genes. Third, epigenetic changes can be mediated through micro-RNA (miRNA) and messenger RNA (mRNA).
Inflammation, Macrophages, and ER Stress
DIABETES-ACCELERATED ATHEROSCLEROSIS AND INFLAMMATION – REVEALING THE PICTURE ONE PIECE AT A TIME
Karin Bornfeldt, PhD (University of Washington, Seattle, WA)
Dr. Karin Bornfeldt reviewed recent basic science research into atherosclerosis progression and diabetes. Her takeaway was clear and unsurprising: diabetes accelerates several of the processes of atherosclerosis. Much of her presentation focused on the role ACSL1 (an enzyme that converts free fatty acids to acyl-CoAs) plays in this process. A plethora of mouse studies have demonstrated that ACSL1 is required for the diabetes-induced inflammatory monocyte and macrophage phenotype and diabetes accelerated atherosclerosis. Dr. Bornfeldt and colleagues have not yet started to evaluate ACSL1 as a drug target, though her team is in the process of identifying other potential drug targets in the same pathway.
- Diabetes promotes accumulation of macrophages in the artery wall. Dr. Bornfeldt showed how mice with diabetes have larger early atherosclerotic lesions due to increases in macrophage accumulation. Encouragingly, she also showed how this effect of diabetes can be prevented by insulin treatment.
-- by Adam Brown, Hannah Deming, Nina Ran, Joseph Shivers, and Kelly Close