Understanding your materials and methods

Office

Imagine you’d perform a clinical study in healthy volunteers to assess the activity of a new anti-inflammatory drug. How to demonstrate the compound’s anti-inflammatory effect in healthy subjects? These subjects don’t have chronic inflammation; they’re healthy so there’s nothing to treat. A solution is to induce inflammation outside the body. This is done by exposing blood from volunteers that received the drug to a foreign trigger. The theory is simple: if the drug has the intended pharmacological activity, it reduces the trigger-induced inflammatory response.

Obviously, this inflammatory response should be completely controlled in terms of magnitude (what’s the cytokine level?) and nature (what is the pathway producing cytokines?). No unintended inflammation may be present in the test tube. For this reason, only blood collection tubes are used that are endotoxin-free. Endotoxin is a bacterial component that is everywhere, contaminating everything that is handled by human hands. This is certainly not something that you’d want to be in your test tubes!

The clinical trial is executed, cytokine analyses are run, and data are reported. Data analysis reveals that the anti-inflammatory drug did not inhibit inflammation at any dose level tested. Moreover, even the blank conditions (without any inflammatory trigger added) show massive cytokine release! A failed clinical study, who to blame?

This is a hypothetical case, but not inconceivable to happen. We discovered that immune cells collected in heparin tubes may become activated, even though the tubes were tested to be endotoxin-free. This activation differed per manufacturer and even per tube batch. Apparently, heparin tubes may contain an unknown trigger activating the immune system. Luckily, we discovered this before the blood collection tubes were used in a clinical study. Hence, we decided to always test each future tube batch for undesired immune stimulation prior to clinical use. Thereby we avoided a failed clinical trial.

The verification of materials and methods may not be the sexiest topic to blog about. However, above example demonstrates the importance of control and understanding of all experimental details in clinical research. Following the text books, the manufacturer’s specifications, or PubMed is not sufficient. Artifacts may be introduced at different levels: preanalytical, analytical and postanalytical variables play a role. An unsuitable blood collection method may activate platelets and interfere in your readouts, a delay in sample handling may result in reduced responsiveness of the cells in your bioassay, your primary readout measure may be subject to diurnal fluctuations, etcetera. Just imagine that we’d not have tested the blood collection tubes preceding our clinical study. The loss of information, time and money would have been enormous!

Matthijs Moerland

Don’t forget to check out CHDR’s new Annual Report! Click here to download the book.

How to test subjects at home

Tuesday morning, 8.55 hours: monthly scientific advisory board meeting at CHDR.

I was one of the first to arrive at CHDR’s auditorium this morning as I will be presenting the new study that I and Christophe, the elective student who is working with me on the project, are preparing. As the auditorium is filling up and most of the regular attendees find a seat my heartbeat slightly increases in anticipation of giving the presentation. It is not that I am not capable of giving an oral presentation, only this time we have incorporated an extra feature into the presentation that will give a live insight into the study that I will be presenting.

While Christophe is checking the functionality of our surprise feature for the last time Piet Hein van der Graaf opens the meeting by welcoming everybody. Four session are on the menu this month: three introductions on new projects and one presentation on the results of a project that was already finished. According to the agenda Christophe and I are up for the second talk so we are still relaxed. However, the presenter of the first study is experiencing some familiar problems with the national railway and will not make it in time for the first talk.

This means that we are up to kick off the meeting. While I gather my papers and find my way to the microphone I start to wonder what effect this unexpected change will have on the surprise feature.

After the general introduction to the topic of the study, at home monitoring of two patient groups using a wearable biosensor, Christophe takes the microphone to address the technical aspects of this so-called healthpatch, which is able to continuously monitor the user’s heart function, respiratory rate and body position.

Another feature present on this biosensor is stress-level detection. And this is where our presentation becomes very interesting. What nobody but Christophe and me knows is that I am wearing the biosensor on my chest and in the next slide the audience will be able to look at my live vital signs during the presentation. The thought of everybody studying my stress level already causes slight stress. As the next slide appears, revealing my live ECG, heart and respiration rate and my stress level and the audience realizes what they are actually looking at people start making jokes about my rising stress level, which is indeed the case. One person even wonders if an ambulance should be called as my heartbeat keeps on rising. Next to the feeling of exposing myself in front of the whole advisory board this nicely reflects the increased stress that many will recognize when standing in front of an audience that is eyeballing you.

An interesting phenomenon is also seen at the end of my talk, during the questions: everybody can see that my heart and respiratory rate increase when a question is asked and just before I answer these vital signs quickly decrease again, a reflection of my initial insecurity on my ability to answer the question and the subsequent relieve of knowing the answer.

Using this device to monitor patients in the comfort of their own homes will reduce burden and costs of clinical trials, as well as provide more naturalistic data as they did for us in a way hitherto impossible. Watch out for my next blog where I hope to be able to share the first results of our trials@home project!

Ellen ‘t Hart

The research clinic. Ready 4 research

Clinical 06- LQ

The development of new drugs also takes long, because clinical trials are difficult to perform – partly because enrolment of patients is slow. This is not because patients are unwilling to participate. CHDR regularly approaches patients with a certain disorder directly, through advertisements in newspapers and on the internet. Often, literally hundreds of interested patients respond to a single campaign. In fact, the direct approach of patients is much more efficient than recruitment via doctors or hospitals. However, usually only a minority of the respondents qualify for the study.

Whereas self-management of diagnostics and therapeutics changes rapidly, patient involvement in clinical trials is lagging behind. Many studies fail because recruitment targets are not met. Multicenter trials where each center contributes a small number of patients hugely increases costs and complexity, and thwarts more sophisticated research. CHDR has therefore started another initiative to facilitate clinical trials: our first Ready-For-Research clinic in psychiatry will open this month, clinics in rheumatology and diabetes will follow soon. CHDR has a good overview which drugs  are developed by pharmaceutical industries. We invite patients with matching diseases who are willing to participate in clinical trials to come to our Ready-For-Research outpatient clinic, not for a concrete protocol but for a full medical screen and inclusion in our research database. With hundreds of respondents to advertisement campaigns, it shouldn’t take long to gather enough patients. The features of this particular group are used to approach pharmaceutical industries, which then allows the design of a protocol that suits the patients who are ready for research. This is more naturalistic than most predefined selection criteria, which often fit only a few percent of the population. Obviously, a patient’s decision to participate is still completely voluntary, and not all individuals will be eligible. But getting patients Ready-For-Research before the study is designed, will be much more efficient than trying to find them only after ethics approval of a protocol that excludes most patients.

CHDR is sharing this initiative with organizations for patients and medical professionals. Some diseases don’t attract much attention from the pharmaceutical industry, because they are rare and studies are considered difficult – even if the industry has a potentially effective drug in development. This may change when enough of those patients are ready to participate in a trial. Drugs that are primarily developed for a more prevalent condition, can then also be effectively studied in a rare disease where they may also have beneficial effects. It may not always be possible to find a study that matches the unmet medical needs of patients with a certain disease. But together we have a much larger chance of accelerating drug development. When patients get Ready-For-Research, investigators can design more efficient protocols. Patient empowerment should also focus on their contributions to clinical research.

Publication: we all benefit

Office 07- LQ small

It is always interesting to see a sponsor’s reaction when we describe the Dutch directive related to publication of trial results. It either ranges from nonchalant shrugging of shoulders to raised eyebrows to utter panic. In the Netherlands, the Competent Authority has issued a directive that the clinical trial agreement cannot include unreasonable restrictions on publication of trial results. Many small biotech companies are very open to publishing their trial’s results. On the other hand, there is an unfounded fear amongst some pharma (or at least their lawyers) that their confidential, proprietary data will be published without their consent. Of course, there are safe-guards in the directive preventing this kind of recklessness. And unsurprisingly, in a collaborative setting this is never the case.

The merits of publishing (and the perils and biases of not publishing) are well known and are not simply restricted to increasing academic knowledge but indirectly benefits subjects, patients and the community as well. Nonetheless, for a niche CRO such as CHDR, publication of peer-reviewed articles is one of the ways we establish our expertise in an particular field. And many of our sponsors initially become familiar with us by finding research we have (co-)authored in the literature or as poster presentations at conferences.

Also CHDR is somewhat unique in that the clinical scientists and research physicians who work as project managers are also PhD candidates. With the goal that within 3 to 5 years they have completed their degree, this also includes publishing 4 to 7 articles. This construct of having PhD candidates working in early phase clinical research unit has the benefit of educating them in the fundamentals of clinical pharmacology and at the same time they gain experience in how pharma and academia works (or doesn’t). This also enables us to address the knowledge gap between academia and industry.

Besides, all of our senior clinical scientists are encouraged to participate in peer-review of manuscripts in their respective fields. With some of our research directors and CEO, being Executive Editors and Editor-in-Chief of the British Journal of Clinical Pharmacology, respectively. This allows our management team to keep up to date with the latest, cutting edge, clinical pharmacology research.

It is motivating to see that even at a regulatory level there is a push for greater transparency. Since the beginning of 2015, the EMA will  proactively publish the clinical reports submitted as part of marketing-authorization applications for human medicines, no doubt including many early phase studies. Similarly, it will be interesting to see how the new European Clinical Trial Regulation will be implemented. The regulation includes the requirement for Phase 1 trials to be registered and summary reports and lay summaries published within one year from the end of the clinical trial. No doubt we will see in due course if this forces the disclosure of commercially confidential information or sets out to do what it intended to do and provide information on innovative (and not-so-innovative) clinical research.

Justin Hay, Senior Clinical Scientist

Forgotten players in glucose homeostasis?

Lab 01C- MQ resized

It is highly likely that you learned at secondary school that the pancreatic hormones insulin and glucagon are important in the regulation of glucose metabolism. However, it is equally likely that during your further education more attention was given to insulin compared to glucagon, which at best got the role of bystander. Funnily enough both hormones were discovered around the same time (insulin in 1921 and glucagon in 1923) as was their role in glucose homeostasis. Insulin got all attention as new revolutionary medicine, possibly because Insulin Dependent Diabetes Mellitus (Type I DM) was such a lethal disease that could now be properly treated. In contrast, glucagon-deficiency was not recognized as a clinical entity let alone as a disease. This is all very understandable, but it remains enigmatic why insulin-based therapies also dominated the treatment of Non-Insulin Dependent Diabetes Mellitus (Type II DM). Especially in view of the observations in the 1960’s that Type II DM is not a disease of insulin deficiency, but a condition of insulin resistance which cannot be overcome by increased endogenous insulin release. Whatever the explanation for this enigma may be, we are currently witnessing a revival in glucagon-based therapies for the treatment of Type II DM.

At CHDR we have adapted and validated existing techniques to investigate the role of glucagon in glucose homeostasis and to evaluate glucagon-based therapies in humans. The development of these tools with our colleagues in industry and academia was and will be a hallmark of CHDR’s philosophy. This also regards the development of mathematical models that try to capture the complex interplay between insulin, glucagon and glucose. We are proud that all this work is now integral available as PhD thesis, which our colleague Marloes van Dongen will publicly defend on January 7, 2015 in the historical Academiegebouw of Leiden University.

An important role of the specialty of clinical pharmacology is to develop the tools of the drug development trade. We sometimes define new (and untested) medicines as extremely sophisticated technological innovation after evaluation with rather primitive methodology. The ongoing drug innovation wave has to be matched with equally sophisticated methodology to see the potential effects. These techniques have to be developed preferably ahead of a new innovation wave in a certain field[1].

Renewed interest in glucagon as one of the ‘forgotten’ players in glucose homeostasis, of which there are more[2], will lead to a potential wave of new products and devices. We are happy that our work is now available to support this, and make a difference in the attack on the Type II DM epidemic.

Koos Burggraaf

[1] Cohen et al. Annu Rev Pharmacol Toxicol. 2014 Oct 6. [Epub ahead of print]; PMID: 25292425

[2] ‘certain factors produced by the intestinal mucosa in response to nutrient ingestion that are capable of stimulating the release of substances from the endocrine pancreas and thereby reducing blood glucose levels’; see for instance Bayliss WM, Starling EH. Proceedings of the Royal Society of London[Biol] 1902;69:352-353 / Moore B, Edie ES, Abram JH. Biochem J 1906;1:28-38) and La Barre J. Bull Acad R Med Belg 1932;12:620-634.

Innovative drugs need innovative methodology!

Figure_CHDR_Study_Setup_myelin_breakdown

In drug development it is important to try to answer as many questions as possible in as few drug studies as possible. If you expose humans –or other animals for that matter- to new and potentially harmful compounds, then not getting everything you can out of it, seems a waste. After a new drug has successfully passed the stage of animal studies, a “first in human” study is planned in which  -traditionally- pharmacokinetics of the new drug and potential side effects of the compound are determined. In this phase, however, it is likely that humans will be exposed to a broader range of drug doses than ever thereafter. An ideal opportunity to also try to determine the intended pharmacological effects of the new compound. To do that you need the correct methodology and this is often unavailable.

Luckily, more and more pharmaceutical and biotechnology companies are seeing the added value of this approach. CHDR’s self-funded research is focused on the development of new biomarkers and methodology to show pharmacological effects of new drugs in the earliest stages of clinical drug development. There is a new trend: co-funding of this type of research by a pharmaceutical company with a particular interest in the new method rather than just the new medicine.

Multiple Sclerosis is caused by inflammation that leads to demyelination -loss of the myelin sheath around neurons- in the brain and spinal cord. Existing therapies target inflammation and thus lead to inhibition of demyelination. However, new MS drugs are being developed, that target enhancement of the formation of new myelin and hence improvement of the function of demyelinated neurons. These targets are completely new and methods to quantify the effects of these innovative drugs therefore don’t yet exist. In collaboration with a biotechnology company, we developed and validated a method that uses labeling with deuterated water to estimate the speed at which new myelin is formed in the central nervous system. Healthy subjects drank 120 mL of “heavy water” per day for a period of 10 weeks and we performed repeated lumbar punctures to obtain cerebrospinal fluid over a period of almost half a year. We then extracted myelin breakdown products from the CSF and measured the rate of weight increase of the molecules, which was caused by incorporation of deuterium instead of hydrogen in newly formed molecules. These measurements, in combination with mathematical modeling, allowed us to estimate the rate of myelin formation.

Being able to quantify the rate of myelin formation will be essential in the process of developing a drug that is expected to positively influence remyelination. This may ultimately lead to a new treatment for patients with MS, which is much needed. But the method by which metabolic processes that occur within the confined space of the brain can be quantified using something as innocuous as water, may in turn contribute to the rational drug development of many other future CNS drugs.

Geert Jan Groeneveld

Cars & Clinical trials: driving simulators Vs. on-the-road-driving-tests

BvkYjb5IMAER8ow

For many of us, driving a car represents freedom and independence and economical power. What has this to do with CHDR, you might ask? Well, many pharmacological agents that affect the central nervous system are of concern when it comes to driving safety. Those medications (and also drugs of abuse) can induce fatigue, impair vision and reduce vigilance leading to periods of inattention. This poses a risk for the operation of dangerous equipment like a car. Although those risks are often already known at a drug-group level, for clinical pharmacologists like us, it is important to estimate the dose response relationship for an individual investigative drug early in its development. Additionally there may be certain groups, like the elderly or the young drivers, which are much more at risk when impaired by medicines, alcohol or drugs.

The effect of investigative drugs on driving performances is often assessed by using a standardized on-the-road-driving-test, which was developed in the 1980s and has been applied extensively in over 50 studies since. In short, subjects are instructed to drive a car over a 100 km highway. The car is equipped with two camera’s that constantly monitor the position of the car within the traffic lane. The outcome is mainly measured by the alterations in the standard deviation of lateral position (SDLP), which has proven to be a suitable parameter for vehicle control and traffic safety.

However, the on-the-road-driving-test is inflexible, labor intensive and therefore costly, it may take weeks to complete a full study while testing a single dose only, making the test less suitable for the assessment of dose response effects and certainly not suitable for a quick adaption in the study design. Nevertheless, the on-the-road-driving-test is often required for registration purposes, but a negative study outcome may cause a significant restriction in its indication as specified in the SMPC. Therefore, an on-the-road-driving-test may not be the single most suitable tool for drug-induced driving risks.

Luckily, the SDLP can also be measured in an in- house driving simulator in a much more practical and reproducible manner. Other parameters that are of importance in driving a car safely -, i.e. alertness, memory, coordination amongst others – can be added effortlessly. Also, driving simulation makes it possible to study the temporal relationship between medication and driving performance and work out the pharmacokinetic dynamic relationships. Of course all tests are surrogate endpoints for the occurrence of accidents but this ‘real’ endpoint can never be assessed. The current generation of driving simulators have, however, been validated positively against real driving behavior.

At CHDR we are currently studying the sensitivity of a driving simulator test battery to the effects of ethanol and a benzodiazepine, trying to compare these results with those of the NeuroCart™, a validated and established test battery developed by CHDR that quantifies a large range of drug-sensitive CNS-functions that are also relevant for every-day performance. Results from this study will allow us to determine whether a driving simulator is suitable to assess impairment in performance due to investigative drugs in a laboratory setting. This will set the standard for future studies in the important area of drug effects on real life performance.

Rob Zuiker