The use of MPD Instrumentation in Pharmacokinetics
ABSTRACT: There is an increasing use of animal models in biology, and mouse is becoming a favorite model animal due to the large number of well defined lines of mice, for which genotype and phenotype is relatively well understood. These involve a number of " knock-out" mice. Because, the mouse is a small animal there is an increasing need for new more sensitive detection methods permitting high resolution pharmacokinetics studies without sacrificing animals. Thus, there is a need for new super-sensitive, instrumentation which advances the application of radiotracer methodologies for pharmacokinetics, and in the future, molecular biology imaging applications, including cell communication and gene expression. MPD enhanced pharmacokinetics studies (PK/MPD) demonstrated the considerable advantages of our supersensitive Multi Photon Detection (MPD) instrumentation.
MPD enabled methods for in Pharmocokinetics: In the case of small animals, such as mice, it is currently difficult to perform uptake/clearance and pharmacokinetics studies without scarificing tens or hundreds of mice. This leads to considerable "political" problems, is costly and leads to measurement artifacts when the mouse is not a pure line. Thus, it is important to provide more sensitive methods which permit studies in which the blood sample is less than 5 microliter, i.e., the mice do not need to be sacrificed. Many uptake/clearance and metabolism studies are performed using radio-iodinated biologicals. However, their use has been limited by the need to use non-negligible (often not physiological) amounts of radiolabeled biologicals and the need to kill the animal. This limitation is mainly due to mediocre sensitivity, i.e. the high background of conventional gamma counters. MPD instrumentation permits the use of much lower amounts of radioactivity and only a few microliters of blood need to be drawn.
Recently, in collaboration with Dr. M. Kubin, Immunex, Seattle, we performed pilot studies on the use of MPD instrumentation in pharmacokinetics. In this experiment a proprietary, singly radioiodinated protein of molecular weight about 75 KDalton was injected into mouse. All mice were injected with the same amount of radiolabeled biologicals and a special adjuvant expected to modify metabolism have been added in half of the cases. We demonstrated that in uptake/clearance studies, MPD instruments permit the use of 200 times less blood, i.e., we can perform 100 time-point measurements using a single animal and this animal does not need to be sacrificed.
In the above described experiment one expected a rapid degradation of the injected protein. The protein was expected to rapidly degrade into individual amino acids, of which only thyrosine would have been iodinated. Thus, we performed precipitation studies in which the intact protein is expected to be in a pellet and the metabolized products in the supernatant. We used the following procedure:
The results are provided in Figure 1. We demonstrated that the use of MPD instrumentation permits studies of the precipitable fraction of radiolabeled biologicals when using only 5 microliters of animal blood. Thus, these studies can be performed without sacrificing the animal.
The measured ratio of precipitable/non-precipitable do not give full characterization of in vivo metabolism and degradation processes. For example, when the protein is cut into two fragments, both are large enough to precipitate. Thus, instead of the precipitation procedure, one should perform 1D gel or capillary electrophoresis separation. Such studies have been rarely performed due to severe technical limitations. First, if all available sample, say 200 microliters, is loaded on a gel, one observes catastrophic overloading artifacts. Actually, our experiments suggest that one can not load more than 10 microliter and loading of 2-3 microliter seems optimal. Second, the sensitivity of prior-art imaging detectors is much lower than of counters. For example, the detection efficiency of a phosphor imager for 125I is about 0.5 % whereas a good gamma counter has a detection efficiency of up to 80%. Finally, the use of controls is handy, but all prior art imaging detectors are unicolor.
The MPD Imagers we developed have been used to provide the necessary sensitivity to analyse the 1D and 2D gels. We performed 1D gels analysis of the serum content subsequent to injection of radioiodinated protein X with Mw = 75 K Dalton. For that, 1 ul of total serum samples were denatured in 1.5% SDS sample buffer with 50 mM DTT. Serum proteins were analyzed in 4-12% SDS-PAGE (NOVEX, CA) followed by imaging the gels on an MPD Imager and analyzing the data with in-house software. We demonstrated that the total amount of radioiodinated protein in 1 microliter of serum is only a few attomole. Thus, only the supersensitive MPD Imager can reliably quantify this amount.
The analysis of the relative efficiency of 1D gel vs. precipitation methods have been performed for four samples. The results show excellent corellation between the estimates of the amount of intact protein obtained from 1D gel and precipitate measurements. However, 1D gel permits assesing the amount of partially digested protein, which in our case is rather low, typically 5-15% .
Summary: We demonstrated that the supersensitivity of MPD instrumentation permits pharmacokinetics studies using only a few microliter of animal serum. Thus, a long series of pharmacokinetics studies can be performed without sacrificing the animal. The method seems to be of significant importance for small animals, e.g., mouse.