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The use of flow cytometry can add value in Phase I studies.
Drug development is a costly process. After passing the pre-clinical phase, the majority of new compounds are still eliminated during the (early) clinical phase. During this early phase, focus on safety of the volunteers is of the utmost importance. Apart from that (from an economic point of view), it is favorable that new compounds with insufficient efficacy or intolerable side effects are identified as early as possible in Phase I. The use of biomarkers is one of the key requirements that allow for proper decision making. The analysis of samples using flow cytometry can provide cell data to support early determination of drug efficacy.
Flow cytometry is increasingly used for safety/exploratory purposes in pre-clinical and clinical trials. Using this technique, size, activation state, or expression of biomarkers of specific cell populations can be quantified relatively easily, since multiple markers (up to 18) can be analyzed simultaneously. From a safety point of view, flow cytometry can be used to assess the effect of a test compound on absolute numbers, or percentages, of multiple leukocyte subsets.
However, flow cytometry can also be used for the development and validation of assays that demonstrate the efficacy of the compound, both in vitro and ex vivo, as early as Phase I, provided that sufficient drug concentrations are reached. Moreover, the obtained flow cytometry results can be used for integration of PK and PD results of the compound. A new concept has been developed to employ validated flow cytometry methods to obtain more information that is valuable for decision making on the fate of new compounds. A few examples will be given below.
Flow cytometry methods can be applied for either small-or large-molecule compounds that interfere with immune cells. Typical examples of these compounds target (chronic) inflammatory processes such as cancer and allergies. First, an in vitro assay in whole blood is developed, in which specific leukocyte populations (e.g., lymphocytes, granulocytes) are stimulated with general stimulatory molecules (chemokines, cytokines, allergens, etc.). Expression of stimulation/activation markers such as transcription factors, adhesion molecules, and intracellular cytokines in this cell population is then analyzed by flow cytometry.
Next, the dose-dependent inhibitory activity of the anti-inflammatory compound in whole blood is assessed in vitro. Care should be taken to make sure that the concentrations of the test compound reflect the concentrations that are expected in the dosed volunteers. During the actual clinical trial, the activity of the anti-inflammatory compound is assessed by evaluating the ex vivo potential of the compound to prevent stimulation in whole blood of dosed volunteers. Obviously, sufficient sampling time points around the expected Cmax of the test compound should be obtained.
Valuable information can be gathered by integrating the PD results, obtained by flow cytometry, with the traditional PK results obtained in the plasma/serum of the volunteers. Moreover, determination of target receptor occupancy by flow cytometry is an important tool to evaluate the pharmacokinetic characteristics of the compound if the test compound is designed to bind to receptors of specific leukocyte subsets. After all, traditional PK analysis is performed in plasma or serum, whereas in these particular cases the compartment that warrants further scrutiny is the specific target cell population.
As all analytical methods, flow cytometry yields both advantages and disadvantages. Flow cytometric methods are fast, quantitative, and provide important information for clinical trials. However, the best results are often obtained with fresh whole blood samples (although it is possible to isolate and store cells in a frozen state). Whole blood stability is often limited, and a close collaboration between clinical test sites and the analytical laboratories is required.
These requirements warrant thoroughly validated methods in order to generate valid results, and some excellent white papers are available that describe the requirements with regard to validated flow cytometry methods. The following parameters should always be validated for flow methods:
Procedures should be in place to prevent changes of critical assay reagent lots during analysis as much as possible and describe how to deal with changes if required. Special care should be taken with regard to maintenance and calibration to track proper functionality of flow cytometers that are used to support clinical trials. Tracking these maintenance and calibration data assures that reliable data were obtained during a clinical study. Furthermore, whole blood stability of the clinical sample should be validated in presence and absence of the compound. Documentation is required to demonstrate that samples were processed and analyzed within the validated time periods, in order to generate valid results for interpretation of compound activity. In previous studies, it has been observed that a particular compound may negatively influence the whole blood stability.
Finally, training and assay capability of technicians who are performing the actual analyses during a clinical trial should be documented, and flow cytometry experts should be available for trouble shooting.
All flow cytometry-related equipment that is used at a bioanalytical lab should be validated according to the current guidelines for GLP. All flow cytometric analyses need to be performed within a GLP-compliant facility.
In summary, flow cytometry is a powerful tool that can be used for determination of drug compound efficacy, as early as in Phase I.
Barry van der Strate, is Senior Project Manager Development, Bioanalytical Laboratory at PRA, 4130 ParkLake Avenue, Suite 400 Raleigh, NC, e-mail: StrateBarryvander@praintl.com.