Metabolic mass balance studies

As discussed in Section 4.2.3, the elimination of 2 -MOE partially modified AS Os is attributed to slow (but continuous) nuclease-mediated metabolism in tissue, followed by ultimate excretion of these shortened metabolites in the urine. This major elimination pathway for 2 -MOE partially modified AS Os is shown graphically in Fig. 4.9, where approximately 75% of the total radiolabeled dose is excreted in the urine by 90 days after a single dose (5 mg/kg) of [3H]-ISIS 104838 to rats [26]. Another example of the ultimate elimination of this class of compound via slow metabolism in tissues, followed by the urinary excretion of metabolites, is the mass-balance study with [3H]-labeled ISIS 113715. Here, between 8% and 37 % of administered dose was excreted within the first 24 h, followed by a daily ur-... 

CYP3A4 metabolizes 1 to four metabolites, whose activities at V]a and V2 receptors are 3-50% and 50-100% that of 1, respectively.28 However, the combined exposure to these metabolites after i.v. administration of 1 is only 7% that of parent, so their contribution to the clinical effect is minimal. A mass-balance study with radiolabeled 1 revealed that 83% of the dose to be eliminated in the feces, with the remainder being eliminated in the urine.28... 

ADME Since metabolism and formation of active metabolites are not a concern for unmodified biopharmaceuticals, mass balance studies are uninformative. Tissue concentration of radioactivity using radioactive proteins is also difficult to interpret due to unstable radiolabel linkage, rapid in vivo catabolism, and recycling of radiolabeled amino acids into non-drug-related proteins/peptides. 

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 distri-... 

Systemic bioavailability is the product of fraction of dose absorbed (/a), fraction of dose escaping gut metabolism (/g), and fraction of dose escaping first-pass metabolism (F ). Permeability class is based upon /a, which may be estimated either in vivo or in vitro by direct measurement of mass transfer across human intestinal epithelium. In vivo methods include (i) mass balance studies using unlabeled, stable-isotope labeled, or a radiolabeled drug substance (ii) oral bioavailability using a reference intravenous dose or (iii) intestinal perfusion studies either in humans or an acceptable animal model. Suitable in vitro methods involve the use of either excised human/animal intestinal tissues or cultured epithelial monolayers. All of these methods are deemed appropriate for drugs whose absorption is controlled by passive mechanisms. 

G. Drug Metabolism Profiling and Mass Balance Studies... 

Permeability can be assessed by pharmacokinetic studies (for example, mass balance studies), or intestinal permeability methods, e.g. intestinal perfusion in humans, animal models, Caco 2 cell lines or other suitable, validated cell lines. In vivo or in situ animal models or in vitro models (cell lines) are only considered appropriate by HHS-FDA for passively transported drugs. It should be noted that all of these measurements assess the fraction absorbed (as opposed to the bioavailability, which can be reduced substantially by first-pass metabolism). 

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... 

The total amount excreted in urine up to time t C4ex ) can be estimated by measuring the total volume of urine and the compound concentration in urine, while the entire excreted amount (Aex J can be estimated using a similar method with the confirmation that no more compound can be detected in the last portion of urine collection. Early PK studies usually do not collect urine that requires use of metabolic cages. Renal CL usually is estimated when a compound proceeds to a lead stage. In a routine metabolite identification or mass balance study, urine is collected and can be used for renal CL estimation. 

Mass Balance Studies. Pharmacokinetic mass balance studies apply unlabeled, stable isotopes or radiolabeled compounds to study the extent of absorption and first-pass metabolism, distribution, and excretion of a given compound. In the microdosing approach, a C-labeled compound is administered to human volunteers at doses from as low as one microgram blood, urine, and fecal samples are collected over time and analyzed for C content by accelerator mass spectroscopy to determine half-life, plasma AUC, and maximal concentration (Cmax)- However, these methods are not very popular even when very low doses of radioactivity are involved. Highly sensitive, and more readily available, tech-niques for separation and analysis (e.g., LC-MS, LC-MS/MS) are frequently used alternatives that enable pharmacokinetic investigations and metabolite profiling of nonradiolabeled compounds. 

Nonclinical ADME studies are used to link the animal pharmacology and toxicology studies to humans. Data obtained from the animal metabolism and excretory pathways of a drug may be useful to design a clinical mass balance study. Once in the development stage, ADME data in animals and humans with radiolabeled materials will reveal the major circulating metabolite(s) and... 

Both of these approaches allow for assessment of systemic absorption by not conducting complete mass balance studies (e.g., expired air to catch absorbed compound metabolized to COj or HjO expired end products). In vivo dermal absorption studies not taking into account other routes of excretion must be interpreted with caution. One extension of this mass balance excretory analysis is to assess dermal absorption by only monitoring the primary excretory route for the compound studied. Dermal bioavailability has been assessed in exhaled breath using real-time ion trap mass spectrometry to track the uptake and ehmination of compounds (e.g., trichloroethylene) from dermal exposure in humans and rats (Poet et al., 2000). A physiologically based pharmacokinetic model can be used to estimate the total bioavailability of compoimds. The same approach was extended to determine the dermal uptake of volatile chemicals imder non-steady-state conditions using real-time breath analysis in rats, monkeys, and humans (Thrall et al., 2000). 

Expired air. For 14C-labeled chemicals, the tracer carbon may be incorporated in vivo into carbon dioxide, a possible metabolic product. Therefore, when the position of the radiolabel indicates the potential for biological instability, a pilot study to collect expired air and monitor its radioactivity content should be conducted prior to initiating a full-scale study. Expired air studies should also be performed in situations where the radiolabel has been postulated to be stable but analyses of urine and feces from the toxicokinetic study fail to yield complete recovery (mass balance) of the dose. 

---