Total radioactivity profile

The first set of information generated from the ADME studies are the overall plasma profiles of total radioactivity (TRA) versus time which is compared to the plasma profile of parent versus time measured by a validated LC/MS/MS assay. For those drugs where the parent is the major component at all time points in plasma, the total radioactivity profile usually parallels the profile of the parent. Metabolie profiles of plasma samples generated at different time points by high-performance liquid chromatography (HPLC) analysis followed by radioactivity and mass spectrometric detection provide exposure-related information for parent and metabolites in humans and animal species. In addition, this profile provides information about metabolites that humans are exposed to and how this compares to exposures in animal species. 

Figure 6.20 (a) LC-MS total ion current of neutral loss 175 and (b) HPLC-radioactivity profile of a rat liver microsomal incubation of radioactive compound A. Reproduced from [37J with permission from Elsevier. 

Bia was the most abundant residue representing 78.3% of the total residues, the eprinomectin Bib 8.3%, tire 24a-hydroxymethyl metabolite 7.4%, whereas the sum of the minor 24a-hydroxy-, 26a-hydroxymethyl-, and 7V-deacetylated metabolites represented less than 1.6%. The metabolism profile also indicated that eprinomectin Bj was the major residue in all edible tissues, the eprinomectin Bib representing 7.2-9.3%, whereas five to seven minor metabolites represented only 1-2% of tire total radioactivity. In muscle, however, the 7V-deacetylated metabolite accounted for as much as 3.9% of the total residues. Eprinomectin Bia was also found to be a major metabolite, accounting for 80-85.6% of the total extractable radioactivity in milk. In this matrix, the 24a-hydroxymethyl metabolite represented less than 2%, Ure N-deacetylated metabolite 0,7-2.5%, whereas the contribution by Ure other metabolites was negligible. 

The relative amounts of syn and anti adducts produced in mouse embryo cells did not vary substantially with DMBA concentration (20). However, we found a dramatic difference in the relative amounts of these adducts when the dose of DMBA applied to mouse skin was varied (jl). Figure 9 shows the HPLC elution profiles for adducts formed at a low dose of 14 nmol [ HJ-DMBA. Peaks A,C and D are present in approximately equal amounts, i.e. 29, 21 and 22% of total radioactivity, respectively. However, at a 100-fold higher dose of 1400 nmol, peak C has increased to 39% while A and D have decreased to 13% and 9%. These results indicate that the formation of syn-bay region dihydrodiol epoxide adducts is favored at high doses. Due to this, the total binding to deoxyadenosine (peaks C and D) also increases with dose and ranges from 27% to 48% of the total DNA binding. 

The biodistribution of plasmid can be determined by measuring the rate of disappearance of radiolabeled DNA from the bloodstream and its accumulation in tissues or by the use of fluorescence microscopy to trace the leakage of dye-labeled plasmids from the vasculature. Pharmacokinetic analysis of in vivo disposition profiles of radiolabeled plasmid provides useful information on the overall distribution characteristics of systemically administered plasmids, with one critical limitation. The radiolabel represents both intact plasmid and its metabolites. The plasma half-life of plasmid is less than 10 min, and hence tissue distribution and pharmacokinetic parameters of plasmid calculated on the basis of total radioactivity are not valid at longer time points. Thus, polymerase chain reaction and Southern-blot analysis are required to establish the time at which the radiolabel is no longer an index of plasmid distribution. 

The total radioactivity minus the parent compound concentration (determined by the bioanalytical method) in a specimen estimates the amount of metabolites present. If the difference is minimal and does not change over time, the extent of metabolism is low. For plasma or serum specimens, a small difference indicates that metabolites are not present in systemic circulation. For bile or urine specimens, high levels of radioactivity suggest a primary route of elimination for the parent and metabolites. For a drug candidate cleared primarily by metabolism, a preliminary metabolite profile in urine and bile can determine the number of potential metabolites. When the level of a metabolite in a matrix is high, attempts to isolate and identify the metabolite can be undertaken. If sufficient quantities are obtained, the metabolite s pharmacologic and toxicologic... 

The two most common drug metabolism studies are mass balance and tissue distribution. Mass balance studies are usually conducted in both the rodent and the nonrodent species used for toxicology evaluations, whereas tissue distribution is performed only in the rodent. For mass balance, a radio-labeled compound is administered to the test species and urine, feces, and, if necessary, expired air are collected at intervals and counted for total radioactivity. Commonly used intervals are 0-4, 4-8, 8-12, 12-24, and then daily, up to 168 hours or until more than 95% of the administered dose has been excreted. Depending on the pharmacokinetic profile of the candidate, other collection intervals can be selected to give a better picture of the excretion profile. For tissue distribution, a radiolabeled compound is administered to the test species, and after predefined times, usually 2, 4, 8, 24, and 48 hours, the test species... 

White et al. [85] also administered I125-labeled bromelain orally to rats. At various times blood was drawn and the total radioactivity, the trichloroacetic acid precipitable I125 compounds, and the molecular weight profiles of the labeled proteins were determined. They found a maximum absorption in the blood, corresponding to 270 ng/mL bromelain, one hour after bromelain administration. Approximately 40% of the radioactivity in plasma could be precipitated by 10% trichloroacetic acid. Electrophoretic analysis showed one major peak of radioactivity in plasma, with a molecular weight of 26,000-30,000 daltons. This maximum peak contained 0.003% of the administered dose of bromelain per mL plasma. 

Following collection, tissue and excreta samples are analyzed for total radioactive content and the pooled samples (by sex and/or sacrifice interval) are processed and analyzed to determine the metabolic profile of tissue and excreta radioactivity. Following profiling, major tissue and excreta metabolites are isolated and identified. 

If little or no previous data on the metabolism of the compound in the target species is available, it is prudent to consider conducting a probe residue depletion study in three animals sacrificed at three widely spaced intervals following the last dose. For example, one animal might be sacrificed at 12 hours (zero withdrawal) and another each at 72 and 120 hours post final dose (the exact times selected will depend on the tissue clearance of the drug under development). The information obtained will allow one to select more accurately sacrifice intervals for the definitive study which encompass proposed safe concentrations of total drug-related residue. Additionally, the probe study residue data will allow for adjustments in the specific activity of the radiolabeled dose to ensure tissue concentrations of total radioactivity at later sacrifice intervals which are sufficient for metabolite profiling and isolation. 

Metabolite Profiles in Liver. The total radioactive residue in all 12 livers was efficiently extracted with a mixture of formic, trifluoroacetic, and hydrochloric acids. After neutralization of the acids with ammonium hydroxide and partial purification by solid phase extraction techniques, HPLC/RAM analysis produced essentially a two-component profile. The minor component was pirlimycin itself in relative amounts of 27.6% 9.7%. The bulk of the residue, 77%, was a single substance and was identified by co-chromatography with an authentic standard and by FAB/MS as pirlimycin sulfoxide (PS). The microbiological analysis of the extracts confirmed this relative ratio since PS has an antibiotic activity 142 times less than that of pirlimycin against M.luteus. The ratio of pirlimycin to total residue was therefore established and will provide the necessary correlation of residue depletion needed for the calculation of residue marker concentration (Rm) and the establishment of a tissue withdrawal period. 

HPLC profiles of the urine extracts from cow 821 and cow 59 are given in Figure 1. There is only partial resolution between peaks 4, 5 and 6. The overall profile has not changed between the two animals except for some variations in the total percentages of the parent compound (peak 8) and the metabolites. There are at most five major (> 10% of the total radioactivity) components in the urine extract. 

FIGURE 9.3 Comparative plasma profiles of radioactivity in rat, dog, and humans showing parent and metabolites after a single oral dose of C-14-labeled compound. The table in the figure shows the percent AUC of total radioactivity for parent (P) and two major circulating metabolites in plasma samples. The AUCs for parent and metabolite were generated from radioactivity profiles generated at several time points. 

Profiles of buspirone metabolites were determined by HPLC with three radiodetection techniques (see Fig. 10.2). Percent distribution of radioactive peaks was calculated by dividing the radioactivity of a metabolite peak by the total radioactivity determined in the HPLC run. ( 8000 DPM per injection for HPLC-LSC and HPLC-MSC, and 32,000 DPM per an injection for HPLC-RCD). 

Radioactivity Recovery Determination Determination of the radioactivity recovery from an HPLC analysis is a necessary procedure to ensure accurate determination of all radioactive components in the original sample. In addition, because metabolite profiling by MSC requires the additional step of in vacuo removal of HPLC solvents, it is also important to determine whether volatile metabolites are lost in the process. We have developed a simple method to determine HPLC column recovery, plate recovery, and the total recovery for an HPLC-MSC analysis (Zhu et al., 2005b). In general, aliquots of a sample are injected onto an HPLC with and without an HPLC column. All effluent from each HPLC run are separately collected and aliquots are analyzed for total radioactivity by LSC with and without solvent evaporation. The DPM values obtained are used to calculate the recoveries. 

Accelerator mass spectrometry (AMS) is an ultrasensitive analytical method for radioactivity analysis. AMS offers 10 -10 -fold increases in sensitivity over LSC or other decay counting methods so that levels as low as 0.0001 DPM can be detected (Brown et al., 2005, 2006). AMS has been applied to mass balance determination, pharmacokinetic studies of total radioactivity, and measurement of chemically modified DNA and proteins in humans after the administration of a low radioisotope dose (approximately lOnCi/person for mass balance and drug metabolism studies) (Buchholz et al., 1999 Garner, 2000 Garner et al., 2002 Liberman et al., 2004 White and Brown, 2004). In addition, off-line HPLC-AMS has been explored for metabolite profiling after... 

The systemic exposure of circulating metabolite is calculated by estimating the area under the curve for a mean radioactivity versus time plot (Fig. 15.7). The fraction of the systemic exposure contributed by a particular metabolite is calculated by dividing the AUC estimated for a particular metabolite by the AUC of the total radioactivity. Alternatively, the metabolite concentration time profile and metabolite concentrations can be constructed using nonradiometric bioanalytical techniques if metabolite standards are available. 

Fig. 4. Double-label gel profiles of isolated mitochondria obtained during petite induction. Log-phase cultures of 55-R5-3C and 1121 were grown on 2% galactose for 5-6 doublings at 18°C (A), or at 28°C in the presence of 3 mg per ml of chloramphenicol (B), and were labeled with [ H]leucine or [ C]leucine in the presence of 200 Mg per ml cycloheximide. After labeling, mitochondria were isolated and the protein was separated on 10% SDS polyacrylamide gels. After electrophoresis, the gels were sliced and counted. The data are normalized to the total radioactivity recovered from the gels after correction for spillover of the two isotopes. (From Weislogel and Butow. )...

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