Chemical stability with non specific binding assay

Chemical stability with non specific binding assay protocol

Test Compound Concentration 1 μM (different concentrations available) DMSO Concentration 0.33 % Time Points 0, 5, 15, 30, 45, 120 min Number of Replicates 2 Incubation Plate Material PTFE (all time points) and polypropylene (2 hr only) Compound Requirements 50 μL of 10 mM solution Analysis method LC-MS/MS Data Delivery % Parent compound remaining at each time point % Parent compound bound to polypropylene compared to PTFE

Oral administration is the route of choice for new drugs. Compounds must be chemically stable at the low pH values observed in the stomach in order for this to be an acceptable route.

Data from the Cloe Screen Chemical Stability assay

A series of compounds, with potential issues with non-specific binding, was screened through the Cloe Screen Chemical Stability assay. The stability was assessed in PTFE at five time points up to 24hr by monitoring the peak area ratio. The peak area ratio was also monitored after incubating the test compound in polypropylene over 24hr. Using this method, the stability of the test compound and the extent of binding to polypropylene as compared to PTFE could be assessed.

Figure 1

Graph showing the peak area ratio (mean ± standard deviation) of a compound following incubation in buffer at pH7.4 in PTFE and polypropylene over 24 hr.

 

Figure 2

Illustration of a compound which exhibits pH dependent degradation over 120 minutes at pH 2.

 

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Questions and answers on chemical stability

Please provide an overview of the Cloe Screen Chemical Stability assay.

The chemical stability screen measures the degradation of a compound by non-enzymatic processes. This may be caused by several mechanisms, the most common being hydrolysis, oxidation, or light-catalysed degradation.

The test compound (final incubation concentration = 1µM) is incubated with buffer (pH 7.4; final DMSO concentration = 0.1%). Samples are removed at 6 different time points into ice-cold methanol. The concentration of test compound is quantified by LC-MS/MS. The percentage of test compound remaining (relative to the 0 min time point) at the individual time points is then reported.

Figure 3

Degradation of test compound
over 120 minutes at pH 2.

 

Why is the chemical stability of the compound important?

Oral administration is the route of choice for new drugs. Compounds must be chemically stable at the low pH values observed in the stomach in order for this to be an acceptable route. Therefore, knowing if a compound is stable at pH 2 is useful information in predicting if oral administration is likely to pose a problem.

Many in vitro screens are performed at physiological pH to mimic the in vivo situation. If the compound is unstable at pH 7.4, samples generated in the in vitro screens may decay rapidly prior to analysis. Situations such as these may be time consuming and expensive to resolve.

There are two main areas of concern. Firstly, if a compound degrades to a toxic degradation product, and secondly, maintaining a therapeutically effective formulation.

The formulation, storage and packaging of drugs may become important for chemically unstable compounds. Altering the pH of the formulation, removing moisture from the formulation, storing at lower temperatures, or protecting the drug from light may all be used as measures to reduce chemical degradation.

Can the chemical stability be investigated at different pH values?

The chemical stability assay can be performed at any pH. Commonly used pH’s include pH2, pH6 and pH10. We can also investigate stability in a range of buffers such as simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) and also at either room temperature or 37ºC.

How do I interpret the data from the chemical stability assay?

Obviously, a very highly unstable compound is problematic and may preclude development. The decision of whether to proceed with a compound may be based on the intended route of administration, the extent of decay, the reason for the instability and whether procedures can be put into place to reduce the decay. Typically, a compound is classed as unstable if the % compound remaining at the 120 minute time point is less than 85% of the 0 minute time point.

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