An admonition when measuring the lipophilicity of radiotracers using counting techniques

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Abstract

Log P measurements are a fundamental physico-chemical parameter and one of the cornerstones of structure–activity relationships in medicinal chemistry. Despite the attractiveness of the method, the use of counting techniques to measure the log P of lipophilic radiotracers is fraught with pitfalls due to the amplifying effects of small quantities of radioactive impurities. For example, a radiotracer with a log P of 4 containing only 0.1% of a radioactive impurity with a log P of −1 will have an apparent log P of 3 if measured using conventional shake-flask partition techniques, counting the radioactivity in each phase. However, pre-washing the radiotracer-containing organic phase with aqueous phase can, in many cases, allay doubts about the validity of such measurements.

Introduction

An assessment of the property of lipophilicity of a compound is one of the most important and fundamental measurements in the area of rational drug design and development. Quantitative structure–activity relationships (QSAR) have been devised which show the importance of lipophilicity in such diverse areas as cell membrane penetration (including skin and the blood–brain barrier), enzyme inhibition, multiple drug resistance, blood protein binding, and receptor affinity (a recent and comprehensive review of the measurement and uses of lipophilicity has been written by Hansch and Leo (Hansch et al., 1995). In the field of in vivo imaging of biological systems using radiotracers, lipophilicity is often used to help predict delivery of the radiotracer to the desired organ e.g. cross the BBB barrier, and to anticipate the rate of clearance from non-target tissues (Eckelman, 1989).

Estimates of lipophilicity arise from essentially three different methods: computational models based most often on summation of molecular fragments (Rekker, 1977); reverse-phase chromatographic measurements; and partition measurements between water or buffer and an organic solvent, most commonly 1-octanol — the so-called “shake-flask method.” While the first method is obviously important for predictions, actual measurements of lipophilicity are preferred for constructing SARs. The shake-flask method remains the golden standard of lipophilicity and extensive databases are available for comparison of results between different laboratories. At equilibrium, the relative amounts in each phase are measured and the results expressed as a coefficient of partition P, or more often its logarithm log P. When measured at physiological pH the terms log7.4 P, or log D, the log of the distribution ratio at pH 7.4, are often used. A variety of techniques including UV-Vis spectroscopy, GC, and HPLC, and radiotracers have been used to measure the amounts in each phase.

A priori we would expect that measurement of the log P of radiotracers would be a simple and accurate process. This application of radiotracers has the advantages of simple counting techniques to measure the concentration of the radiotracer in each phase accurately and quickly, and as the radiotracer need be present in only very small concentrations, artifacts such as molecular association would be absent, i.e. solutions would be near ideal. However, both anecdotal and published evidence indicate that with radiotracers, measurements of log P have large errors. To quote Hansch and Leo: “It should be noted that at this time we put very little confidence in shake-flask values measured by radiotracer techniques. For reasons not entirely evident, these values (most often for very lipophilic solutes) are very frequently as much as 3.0 log units below those values measured by other methods” (Hansch et al., 1995). We hypothesized that the errors involved in these measurements stemmed from the presence of small quantities of hydrophilic impurities in the radiotracer which significantly skewed the measurements. Many assays of radiochemical purity of a radiotracer would not be able to detect impurities at the 0.1% or even the 0.5% level. Even when such levels of impurities are detected, they are often tolerated because such minor amounts of impurities have no significant effect in the end use of the radiotracer. However, we demonstrate here that for even moderately lipophilic radiotracers (log P > 1), such low levels of impurities lead to large errors in the values of log P obtained by the shake-flask method. We also show that, in many cases, the errors can be eliminated or significantly reduced by a prewash of the organic phase with aqueous buffer before establishing the partitioning equilibrium.

Section snippets

Radiotracer synthesis

The radiotracers used (Table 1) were synthesised from either [11C]-CO2 or [18F]-fluoride produced by a Scanditronix MC 17 cyclotron. With the exception of [18F]-FDG, all were purified by reverse-phase HPLC and formulated in buffered saline at pH 7–8. All radiotracers, with the exception of YF-476 (Semple et al., 1997), are in human use as PET radiopharmaceuticals. All radiotracers were >98% radiochemically pure by HPLC analysis.

Calculations

Let p be the true partition coefficient of the pure compound and q

Results and discussion

Fig. 1 shows two examples of the calculated effects (Eq. (1)) of the presence of small quantities of radioactive impurities of varying lipophilicity in two hypothetical radiotracers which have different log P values. It is apparent that if the impurities have significantly lower log P values than the radiotracer then their effect on the measured log P is enormous, e.g. for a radiotracer with log P of 4, a 0.1% impurity with log P of −1 results in an apparent log P of 3, a factor of 10 or one

Acknowledgements

This work was financially supported by the Medical Research Council of Canada (Grant # MA-14711).

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