Understanding your materials and methods

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

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Publication: we all benefit

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?

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.

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

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

Clinical trials now and then: 1983 vs 2014

Clinical 04- MQIn 1983 I administered my first new medicine to a human being in the department of clinical pharmacology at the Wellcome Research laboratories in Beckenham, UK. It was the antiepileptic lamotrigine (Lamictal) and it eventually reached a turnover of more than a billion pounds, but we were not so sure then. Lamotrigine killed dogs (due to a metabolite that we later found was only formed in the dog) and did nasty things to the kidneys of a certain species of rat.  After internal discussion we went ahead anyway probably to the benefit of many patients with epilepsy and depression, who are currently being treated with lamotrigine. This first in human study was also one of the first with an approval of an ethics committee. Before this we just went ahead after approval by one of the directors.

So the study went ahead. My boss came down from a meeting, had an iv cannula inserted and took the first capsule, returned to his meeting and occasionally walked down for an ECG and a blood sample. The second subject was the chemist who invented the molecule and was rather nervous about causing problems for ‘his’ molecule. This almost inevitably induced a nasty migraine attack. “Don’t winge Dave”, we said as he lay there with a bad headache and we injected anti vomiting drugs in him. The rest is history.

This story may cause severe distress in the modern quality assurance officer, trial monitor, or an attentive regulator. However it is real and by describing the past tries to highlight the contrast with the present.

When we started CHDR in 1987 we very quickly started to do studies for industry and our maiden first-in-human study was with an antiarrhythmic that prolonged the QT interval in the ECG. This cost about 80.000 Dutch Guilders and this would be in today’s terms about the same amount in euros. The protocol had 20 pages and the whole submission dossier to the ethics committee about 30.  The study was completed very quickly after the protocol was written and monitoring was done by one of the employees of the company. She visited us once or twice from Belgium and as a rule brought Belgian chocolates.

The real cost of such a study today is a factor 10 higher, the dossier would be about 20 times more pages and the staff involved in such a study is about tenfold. Of course this is excluding the internal cost of the company. Most likely they will employ an IT company for the study database, an ECG company to read the ECG’s, a central lab company to study the clinical chemistry and a monitoring company to meticulously go over all the blood pressures and side effects. Rules against conflict of interest have done away with the Belgian chocolates. Many of this is global so there is quite a CO2 footprint associated with such a study.

Some of this is good and I would not suggest that we go back to the informal studies of the last century. However, we have a duty to keep new medicines affordable to the whole world. The cost of current drug development is without a doubt unsustainable and the efficiency of the process must be increased. Innovating drug development and its methodology can do this. This is what we have been doing at CHDR for the past 27 years. This new blog series will be written by all our staff members and tell you about our views on this innovation and what we do about it. We hope you are going to read them and share your comments.

By: Adam Cohen