Radiolabeled parent drug

When working with non-radiolabeled drugs the major challenge is to find metabolites in the biological matrices. Because the enzymes responsible for metabolism are quite well characterized metabolic changes can partially be predicted. For example hydroxylation of the parent drug is in many cases the principal metabolic pathway. From a mass spectrometric point of view it results in an increase of 16 units in the mass spectrum. In the full-scan mode an extracted ion current profile can be used to screen for potential metabolites. In a second step a product ion spectrum is recorded for structural interpretation. Ideally, one would like to obtain relative molecular mass information and the corresponding product ion spectrum in the same LC-MS run. This information can be obtained by data dependant acquisition (DDA), as illustrated in Fig. 1.39. 

For a number of years following the discovery and initial clinical use of vinblastine and vincristine, there was relatively little definitive information about the pharmacokinetics of these compounds. Pharmacokinetic studies were accomplished typically using radiolabeled drugs and procedures that were of limited value in distinguishing parent drugs from putative metabolites. 

After oral administration of radiolabeled tilmicosin to broilers at dosage in tire range 25-450 mg/L in water for 3-5 days, radioactivity was mainly distributed to liver and kidney and, to a lesser extent, to muscle and fat. The parent drug was the main residue in tissues, excreta, and bile, but partly desmethylated, hy-droxylated, reduced, and sulfated metabolites could be also identified. Similar pharmacokinetic characteristics were also observed in cattle, swine, and sheep. In broilers treated with tilmicosin at the recommended dosage, residues of the parent drug in liver were 2.6 ppm at day 3 declining to 0.13 ppm at day 17 residue levels in kidney averaged 0.65 ppm at day 3 and declined via 0.08 ppm on day 10 to below 0.06 ppm thereafter. Residues in muscle, fat, and skin were approximately 0.10 ppm at day 3 and less than 0.014 ppm after day 14. 

The metabolism of danofloxacin does not differ in swine. When five daily intramuscular injections of 1.25 mg radiolabeled danofloxacin/kg bw were given to pigs, the parent drug accounted for 72-81 % of the radioactivity excreted in feces and urine over the 5- day dosing period (143). In feces, 5-7% of the radioactivity was identified as A-desmethyl danofloxacin. In urine, 2-3% was A-desmethyl danofloxacin, 10-14% danofloxacin-A-oxide, and 3% danofloxacin glucuronide. 

In sheep orally dosed with 40 mg/kg bw radiolabeled thiophanate, only the parent drug and its major metabolite lobendazole could be detected in plasma for 65 h after dosing. In sheep liver, thiophanate was metabolized to lobendazole at a rate of approximately 34%. Other metabolites included 2-aminobenzimidaz-ole, low molecular-weight aliphatic acids, and limited amounts of the glucuronide and sulfate conjugates. 

Literature data on the depletion of levamisole residues from edible animal products concern only the parent drug (33). It appears, however, that the metabolism of levamisole in food-producing animals is qualitatively similar to that in rats, since limited data from swine (34) and goats (35) are generally consistent with those observed for rats. Metabolism studies in rats using radiolabeled levamisole showed extensive metabolism of levamisole, with at least 50 metabolites identified in some samples of urine from treated rats. 

Metabolism studies in sheep with radiolabeled closantel showed that the parent drug accounted for nearly all the radioactivity in muscle, fat, and kidney. In contrast to tissues in which no metabolism occurred, liver contained two closantel metabolites, 3-monoiodoclosantel and 5-monoiodoclosantel, besides the parent drug. The same metabolites were also identified in feces, although 80-90% of the total radioactivity was due to the parent drug. While amide hydrolysis would also appear to be an alternative metabolism pathway, metabolites that would result from this pathway, such as 3,5-diiodosalicylic acid, have not yet been identified. It might well be that steric hindrance around the amide bonds prevents their hydrolysis (42). 

After a single oral administration of 0.4 mg radiolabeled moxidectin/kg bw to horses, a mean peak serum concentration of 0.134 ppm moxidectin equivalents was attained at 6 h postdose (63). Oral availability was estimated at 40%, while the terminal elimination half-life was approximately 80 h. Within 168 h, 77% of the total radioactivity was excreted mostly by Ure fecal route. In feces, the parent drug represented approximately 70% of the fecal radioactivity, whereas a fraction of 0.28-3.45% was due to four minor metabolites resulting from oxidation mainly on Ci4, C24, and/or C28 positions. 

In swine given radiolabeled ronidazole at the normal feed level for 3 days, total residue concentrations in muscle, kidney, liver, and fat were 8.6, 12.3, 11.9, and 2.5 ppm ronidazole equivalents, respectively, at 0 withdrawal. Total residues persisted in edible tissues by 42 days of withdrawal, at which time muscle contained 130 ppb, whereas liver, kidney, and fat contained 50-60 ppb. Nevertheless, the concentration of the parent drug in the edible tissues was less than 2 ppb at 2 days withdrawal. Ring-intact metabolites including 2-hydroxymethyl-l-methyl-5-nitroimidazole, 1-methyl-2-hydroxymethyl-5-acetamidoimidazole, and 1-methyl-2-carbamoyloxymethyl-5-acetamidoimidazole were found to constitute part of the total residues monitored in the tissues of both animal species. 

Following intramuscular administration of 3.5 mg radiolabeled diminazene/ kg bw in cattle, the metabolites p-aminobenzamidine andp-aminobenzamide were found in the urine besides the parent drug these metabolites constituted 22% and 4% of the total radioactivity, respectively. Liver, kidney, and muscle tissues were found to contain total residue levels of 75, 55, and 2.5 ppm diminazene equivalents, respectively, at 7 days postdosing, declining to 24, 12, and 1 ppm, respectively, at 20 day postdosing. 

Metabolism studies in swine with radiolabeled olaquindox showed that the drug was rapidly absorbed from the gut, more than 90% of the dose being excreted in urine within 48 h after administration (18). In urine, the parent drug constituted more than 60% of the original dose, whereas the remainder was due to five metabolites identified as metabolites n. III, IV, V, and VI. Less than 0.1% of the dose was excreted in the feces within 48 h. 

Pharmacokinetic data with radiolabeled melengestrol acetate showed that the parent compound and/or its metabolites are primarily eliminated with the feces (16). At 6 h postdosing, total radioactivity in heifer liver, fat, kidney, and muscle tissues was 9-15 ppb, 7-8 ppb, 1.2-1.8 ppb, and 0.5-1 ppb of melengestrol acetate equivalents, respectively. In fat, most of this radioactivity (80%) was found to be due to the parent drug, while in liver, kidney, and muscle tissues the parent drug represented about 37%, 30%, and 45% of the total residues, respectively. 

Following administration to rat, dog, rabbit, and cow, clenbuterol was rapidly eliminated, being largely excreted in urine in the form of the parent drug (15). Following a 4 day treatment of cattle at the therapeutic dosage (0.8 g/kg bw) and a 7 day withdrawal, concentrations of clenbuterol in liver were at the level of 0.35 ppb or below, whereas concentrations in urine were approximately one-tenth of the levels in liver (16). Administration, on the other hand, of a single oral dose of radiolabeled clenbuterol to cattle showed that 40% of the urinary radioactivity was due to the parent compound. The urinary half-life of clenbuterol in cattle, estimated from the urinary excretion of the parent compound, was approximately 36 h (17). 

Following oral administration of radiolabeled furosemide, excretion was reported to be almost complete within 3 days in rats (96-98%) and dogs (98-99%). Rat urine contained 40-50% of the parent drug, 30% 4-chloro-5-sulfamoyl-anthranilic acid, and four unidentified metabolites that accounted for the rest of the administered radioactivity. In contrast, urine of dog and monkey contained 85% unmetabolized furosemide, 7% 4-chloro-5-sulfamoyl-anthranilic acid, and the remainder was due to unidentified metabolites. Following intramuscular injection of 5 mg furosemide/kg bw in cattle, the half-life for plasma elimination was estimated at 4.3 h. In contrast, the half-life of furosemide in cattle was reported to be less than 1 h following intravenous administration. 

When channel catfish were intravascularly dosed with radiolabeled acriflavine or proflavine, total residue equivalent concentrations were highest in the excretory organs and lowest in muscle, fat, and plasma (84). In proflavine-dosed fish, residues in liver and trunk kidney were composed primarily of glucuronosyl and acetyl conjugates of proflavine residues in muscle were composed mostly of the parent drug. In acriflavine-dosed fish, the parent compound made up 90% of the total residues in all tissues examined. 

Intravenous administration followed by intramuscular administration 24 h later of radiolabeled tolfenamic acid in dairy cattle at a dosage of 4 mg/kg bw or two intramuscular administrations of 2 mg/kg bw showed tliat at 8 days after the cessation of treatment, the concentrations of residues in liver, kidney, and injection site were 0.07, 0.09, and 39.6 ppm tolfenamic acid equivalent. The proportion of the parent drug relative to total residues was 51% in liver, 56.7% in kidney, and 78% at the injection site. Residues of tolfenamic acid could not be detected in milk at 24-h, following intravenous and intramuscular administrations. 

The amount of total residues is generally determined by study with radiolabeled drugs and is expressed as the parent drug equivalent in milligrams per kilogram of the food. Bound metabolites can be measured as the difference between the total and extractable residue. Microbiological assays measure the parent molecule and its bioactive metabolites immunochemical assays measure the parent molecule and closely chemically related metabolites. 

Excretion concerns the removal of the drug compound from the body. Both the original (parent) drug compound and its metabolites can be excreted. The primary mode of investigation here is excretion balance studies. Radiolabeled drug compound is administered and radioactivity is then measured from excretion sites (e.g., urine, feces, expired air). These studies provide information on which organs are involved in excretion and the time course of excretion. 

Some possible criticisms of the use of radiotracers for monitoring the uptake and release of cocaine are that the tracer chemically reacts with the hair matrix or contains radiolabeled impurities which show preferential binding. Both criticisms are unlikely because (1) the radiotracer never exceeds 1% of the urdabeled drug, and (2) hair exposed to drugs without the radiotracer and analyzed by GC/MS show the parent drug present in amounts indicated by the radiotracer analysis. 

The plasma exposure of a parent drug and major metabolites in the main toxicological species along with any related interspecies differences are of special interest for interpretation of toxicity studies. It is, however, only the data from the human ADME study with radiolabeled material that allows the final establishment of safety margins for all the relevant metabolites, as only then the systemic availability of all metabolites formed in human, is then known. Special attention has to be drawn to major human metabolites, which account for a considerable amount of the exposure (AUC) relative to the parent drug, and human-specific metabolites (Baillie [26]). For the human-specific metabolites and major metabolites that do not reach comparable systemic exposure in at least one animal species used in the different toxicity studies, separate toxicity studies should also be considered [26]. 

As the compound reaches the late discovery and candidate selection stage, the focus is to determine its major metabolic pathways, metabolic difference between species, and to identify potential pharmacologically active or toxic metabolites. Because of the complexity, comprehensive metabolite characterization studies have been typically conducted at this stage with radiolabeled standard. Identification of circulating metabolites is also important at this stage to explain the pharmacokinetic or the pharmacodynamic profile. An NCE may show efficacy that is inconsistent with what is predicted based upon the known concentration of the parent drug. These inconsistencies could be due to the presence of active metabolites. The knowledge of these metabolites will also dictate how the analysis of samples will be conducted in the development and clinical studies. 

---