The Importance of Radiochemical Analysis of Biological Fluids Before and After Lyophilization From Animals Dosed with [3H]-labeled Compounds in Drug Discovery
The Importance of Radiochemical Analysis of Biological Fluids Before and After Lyophilization From Animals Dosed with [3H]-labeled Compounds in Drug Discovery
January/February 2004
Hong Kim, Dan Prelusky, Ph.D. Lydia Wang, David Hesk, Ph.D., Jairam Palamanda, Ph.D. and Amin A. Nomeir, Ph.D.
Departments of Drug Metabolism and Pharmacokinetics and Chemical Research, Schering-Plough Research Institute
Radiolabeled compounds are essential tools in drug research, in particular 3H- and 14C-labeled compounds are crucial for metabolism studies in both drug discovery and development. Compared with 14C, 3H-labeled compounds are not ideal for use in metabolism studies because of loss of the label due to tritium exchange and/or metabolism. However, synthesis of 3H-labeled compounds is easier and faster, and produces higher specific activity than that typically obtained with 14C. 3H-NMR spectroscopy provides a rapid and reliable method for assessing labeling specificity [1]. Therefore, in early and late stage drug discovery programs, 3H-labeled compounds are often used to establish an early direction for synthetic chemists and provide a preliminary account regarding the metabolism and disposition characteristics of the lead compounds. However, for 3H-labeled compounds, caution must be exercised when interpreting the results, and additional experiments may be necessary to avoid misleading conclusions.
Lyophilization is an important technique that is often utilized in drug metabolism studies. Lyophilization is accomplished by freezing the aqueous sample to a minimum of -40?C and then moisture is removed by sublimation under reduced pressure [2,3]. In drug metabolism studies, lyophilization is often used to dry tissues and tissue extracts or concentrate the final products in aqueous media during the isolation of metabolites from biological fluid [4].
In this report, some of the issues associated with the use of 3H-labeled compounds in drug metabolism studies are discussed as well as ways to address these issues. In particular, the use of lyophilization to expel tritiated water (THO) that may result from metabolism and/or tritium exchange of 3H-labeled compound is discussed. Furthermore, several methods for the synthesis of 3H-labeled compounds are described.
Synthesis of 3H-labeled Compounds
Advances in automation, combinational chemistry and high-throughput screening have resulted in increased demand to evaluate a large number of compounds for drug metabolism and pharmacokinetics attributes in early drug discovery. These attributes are usually evaluated using multiple in vitro screens followed by pharmacokinetics in animal models [5,6]. Also, in the current drug development environment, where the success rate of candidate drugs is a major problem for pharmaceutical companies, acceptable drug metabolism and pharmacokinetic attributes of candidate drugs prior to recommendation for development is becoming a must [7].
Selection of a lead compound for progression to development triggers its tritium labeling for the characterization of its absorption, distribution, metabolism and elimination (ADME). In addition, 3H-labeled compounds are often prepared earlier in the discovery program to help, assist and resolve pharmacokinetic (PK) issues that may have arisen. 3H-Labeled compounds are generally prepared by catalytic hydrogen isotope exchange chemistry [8]. Three labeling methods have been employed in the majority of cases, but other methodologies have also been used in a few instances. The simplest method used involves direct 3H exchange of the target molecule using freshly prepared platinum metal and THO. A substrate, a catalyst, a co-solvent and THO are typically heated at high temperature (110?C to 160?C) for as little as one day to as long as one week. The method is effective in labeling compounds that contain a monosubstituted aromatic ring [9].
A second direct method involves the use of a homogeneous Group VIII metal catalyst to exchange tritium via cyclometalation with an appropriate directing group. Examples include rhodium trichloride with THO, and Crabtree's catalyst with 3H2. Reactions are typically complete after an overnight reaction [10].
In many cases, direct exchange of target molecule is not possible and it becomes necessary to label an advanced intermediate. Many molecules contain piperidine or piperazine groups. These can be efficiently labeled using tris-triphenylphosphine ruthenium (II) chloride as catalyst with THO [11]. Reactions are carried out at about 120?C and are complete within 4 hours. After a simple purification, the amine can be converted to the target compound using standard medicinal chemistry routes.
Purification of 3H-labeled compounds prepared by any of the described methods is usually accomplished by reversed phase HPLC. Radiochemical purity, as determined by HPLC with radioactivity flow detector, is typically in the range of 97-99%. In all cases, the site of 3H labeling is confirmed by 3H NMR spectroscopy.
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