At CHDR we can study the effects of immunologically active compounds in vitro, ex vivo and in vivo. In human subjects, in addition to performing routine laboratory tests, we can also use advanced molecular techniques such as immunophenotyping, cell sorting, and ex vivo culture systems. In each context, we can study the compound’s effects on basic physiology, or we can use a challenge model to induce an immune response and then measure the compound’s effect on that response. We are actively involved in developing immunologically active drugs with a wide range of therapeutic applications, including haematology, dermatology, neurology, oncology, rheumatology, and pulmonology. Our robust in vivo challenge model can be either systemic or topical. In some cases, a new preparation may have unintended immunological effects. We have extensive experience analysing the immune response in order to determine the cause of these unintended effects.
Ex vivo challenge models
CHDR has developed an extensive set of ex vivo challenges, which we now use to measure the effect of compounds on a wide range of targets and pathways (see the table below). Moreover, we can develop new ex vivo challenges that are tailor-made to study the mechanism of action of a new test compound.
CHDR has developed LPS challenges for in vitro, ex vivo and in vivo settings.
With the in vitro LPS challenge, we can activate and measure inflammatory cellular pathways downstream of Toll-like receptor 4 (TLR4) using blood samples obtained from healthy volunteers.
Our in vivo LPS challenges (intravenous and intradermal) can be used to safely obtain valuable information regarding the effects of anti-inflammatory compounds in healthy subjects. It can also be used to study specific aspects of the inflammatory response, including the vascular endothelium.
The keyhole limpet haemocyanin (KLH) challenge model
CHDR’s keyhole limpet haemocyanin (KLH) challenge model holds promise for evaluating the pharmacological activity of immunomodulatory agents, already at the earliest stages of clinical development. Developed in-house in 2016, this model has been applied in a number of trials, demonstrating its ability to detect pharmacodynamic effects of novel compounds, as well as its safety and the absence of systemic adverse effects.
Measurement of the histamine-induced skin reaction is a well-established method for assessing the potency and duration of action of compounds such as histamine receptor antagonists or anti-inflammatory drugs. In the histamine skin prick test, four increasing doses (0.1, 0.4, 1.6, and 6.4 μg) of histamine are injected intradermally into the back, using an automatic intradermal injector. Intradermal injection of histamine results in the induction of an allergic skin response (the wheal-and-flare reaction). A clear dose-response is observed when administering histamine intradermally.
Showcase: using the imiquimod challenge to study the effects of TLR7 inhibitors
To evaluate new immunomodulatory therapies in healthy volunteers, we developed a model based on the pro-inflammatory effects of imiquimod on the skin. Imiquimod (Aldara®) is a topical treatment for genital warts, superficial basal cell carcinoma, and actinic keratosis. Imiquimod activates the body’s innate immune system via Toll-like receptor 7 (TLR7), a signalling protein involved in recognising pathogens. CHDR researchers systematically examined the effects of applying increasing doses of imiquimod to the skin of healthy volunteers. Specifically, we studied the cytokine/chemokine cascades that were activated by imiquimod, as well as any resulting histological changes. Importantly, we also found that topical imiquimod does not reach clinically relevant systemic levels, even at the highest dose tested. These results provided proof-of-concept that the imiquimod challenge model is safe, well-tolerated, and fully reversible. Interestingly, the changes induced by imiquimod share several key biochemical characteristics with psoriasis, and CHDR is now using this model to investigate the effects of an immunomodulatory compound designed to treat inflammatory skin conditions.
Showcase: combining ex vivo and in vivo assays during the development of a new TLR4 inhibitor
Ex vivo challenges such as the LPS challenge (see above) provide a safe way to study the pharmacological effects of compounds designed to modify the immune system. Sometimes, however, it may become necessary to validate the ex vivo response using an in vivo approach. This is particularly true when developing a new drug class. In other words, in addition to inducing an inflammatory response in isolated blood cells, it may also be necessary to induce a systemic inflammatory response in human subjects. This is the approach that CHDR researchers used to evaluate the first inhibitor of Toll-like receptor 4 (TLR4). TLR4 activates a variety of pathways that play a role in many acute and chronic inflammatory responses to tissue damage. Thus, an effective TLR4 inhibitor might be useful in treating rheumatoid arthritis and other autoimmune diseases.
Building on ex vivo data, we developed an LPS dosing regimen that triggered a relatively mild – yet reproducible and measurable – inflammatory response in our subjects, providing an in vivo setting in which we could examine the compound’s anti-inflammatory properties. Based on these data, as well as PK/PD modelling and simulations, we were able to determine an effective dosing schedule for studying the compound’s effects in patients with acute or chronic inflammatory diseases. Importantly, this in vivo study also helped us validate our ex vivo LPS challenge, showing that the ex vivo assay can provide a wealth of information without the need to administer LPS to subjects.
Practical answers to important research questions
Does our immunomodulatory compound reach its molecular target, and does it have the desired effect?
Evaluating the pharmacological activity of a new immunomodulatory compound in early clinical testing is not always straightforward. For example, the specific physiological pathway that the compound is designed to target may not be sufficiently active in healthy volunteers. CHDR has the knowledge, resources, and facilities needed to induce and monitor the targeted pathway in in vivo and ex vivo systems. Using this approach, it is possible to quantify the drug’s effects in the earliest clinical stages, showing whether an immunologically active compound reaches its designated target in healthy volunteers.
What is the optimal dose for testing our compound in patients?
We have developed — and continue to develop — both in vivo and ex vivo challenge models that can be used to measure the putative effect of immunologically active compounds on various pathophysiological pathways. Using healthy volunteers, we can now use these robust models in the early phases of drug development to examine the correlation between drug concentration and the intended drug effect. The results of this early testing can then be used to better predict the ideal dosage for use in patients.
Does our biotherapeutic have any unintended side effects?
To investigate whether a new biotherapeutic compound has any unintended immunostimulatory properties (due either to impurities or its inherent mechanism of action), we have developed ex vivo incubation assays using human immune cells. Thanks to our extensive network, we have continuous access to fresh biological samples from both healthy volunteers and patients.