Autoradiography techniques and quantification of drug distribution.

The use of radiolabeled drug compounds offers the most efficient way to quantify the amount of drug and/or drug-derived metabolites in biological samples. Autoradiography is a technique using X- ray film, phosphor imaging plates, beta imaging systems, or photo-nuclear emulsion to visualize molecules or fragments of molecules that have been radioactively labeled, and it has been used to quantify and localize drugs in tissues and cells for decades. Quantitative whole-body autoradiography or autoradioluminography (QWBA) using phosphor imaging technology has revolutionized the conduct of drug distribution studies by providing high resolution images of the spatial distribution and matching tissue concentrations of drug-related radioactivity throughout the body of laboratory animals. This provides tissue-specific pharmacokinetic (PK) compartmental analysis which has been useful in toxicology, pharmacology, and drug disposition/patterns, and to predict human exposure to drugs and metabolites, and also radioactivity, when a human radiolabeled drug study is necessary. Microautoradiography (MARG) is another autoradiographic technique that qualitatively resolves the localization of radiolabeled compounds to the cellular level in a histological preparation. There are several examples in the literature of investigators attempting to obtain drug concentration data from MARG samples; however, there are technical issues which make that problematic. These issues will be discussed. This review will present a synopsis of both techniques and examples of how they have been used for drug research in recent years.

Use of radioactive compounds and autoradiography to determine drug tissue distribution.

Radioactivity has been used in drug discovery and development for several decades because it offers researchers a highly sensitive way to quantitatively assess the absorption, distribution, metabolism, and/or excretion (ADME) of chemical entities by incorporating a radioactive isotope into the structure of the drug molecule. Regulatory agencies around the world require drug makers to characterize the ADME properties of prospective new drugs as one way to help ensure that patients are not exposed to dangerous drug and/or drug metabolite levels before they can be approved for human use. Radiolabeled compounds have consistently proved to be the most efficient tool for determining that information, even though attempts have been made to use nonradioactive techniques. The techniques of quantitative whole-body autoradiography (QWBA) and microautoradiography (MARG), which rely on the use of radiolabeled drugs, are two techniques that are routinely used to examine tissue distribution of drugs in discovery and development. These techniques provide drug researchers with quantitative tissue concentration data and a visual location of those concentrations in intact organs, tissues, and cells of laboratory animals. It is important for readers to realize that these techniques visualize total radioactivity, which can include the parent molecule along with its metabolites, and/or degradation products or impurities. This requires investigators to treat the quantitative data with caution unless the identity of the radioactivity is determined using some type of other bioanalytical techniques, such as mass spectroscopy and/or radio-HPLC, which can be easily performed on the tissue obtained from the animals used for QWBA and/or MARG. Nevertheless, these data are used in drug discovery and development to answer questions related to tissue penetration, fetal/placental transfer, tissue retention, routes of elimination, drug-drug interactions, enzyme induction/inhibition, formulation comparisons, in vivo compound solubility, differential metabolite distribution, interspecies comparisons, and to predict human exposure to parent drugs, metabolites, and radiation during clinical studies. This review will consider the strategic use of WBA, QWBA, and MARG in the pharmaceutical industry. Case studies and anecdotal information will also be presented; however, readers should realize that these are general examples and that some details have been omitted for brevity and/or because the data is proprietary and could not be presented at this time. Nevertheless, the images and discussions are provided to demonstrate how the techniques can and have been used to examine in situ tissue distribution of therapeutic compounds.

Autoradiography, MALDI-MS, and SIMS-MS imaging in pharmaceutical discovery and development.

Whole-body autoradiography ((WBA) or quantitative WBA (QWBA)), microautoradiography (MARG), matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), and secondary ion mass spectrometric imaging (SIMS-MSI) are high-resolution, molecular imaging techniques used to study the tissue distribution of radiolabeled and nonlabeled compounds in ex vivo, in situ biological samples. WBA, which is the imaging of the whole-body of lab animals, and/or their organ systems; and MARG, which provides information on the localization of radioactivity in histological preparations and at the cellular level, are used to support drug discovery and development efforts. These studies enable the conduct of human radiolabeled metabolite studies and have provided pharmaceutical scientists with a high resolution and quantitative method of accessing tissue distribution. MALDI-MSI is a mass spectrometric imaging technique capable of label-free and simultaneous determination of the identity and distribution of xenobiotics and their metabolites as well as endogenous substances in biological samples. This makes it an interesting extension to WBA and MARG, eliminating the need for radiochemistry and providing molecular specific information. SIMS-MSI offers a complementary method to MALDI-MSI for the acquisition of images with higher spatial resolution directly from biological specimens. Although traditionally used for the analysis of surface films and polymers, SIMS has been used successfully for the study of biological tissues and cell types, thus enabling the acquisition of images at submicrometer resolution with a minimum of samples preparation.

Distribution of radioactivity in bone and related structures following administration of [14C]dalbavancin to New Zealand white rabbits.

Penetration of dalbavancin into noninfected bone and joint tissues was assessed after an intravenous dose of 20 mg/kg (of body weight) [(14)C]dalbavancin given to rabbits. Drug-derived radioactivity, determined over 14 days by either liquid scintillation counting or autoradiography, remained above the MIC for common gram-positive pathogens that cause bone and joint infections.

Autoradiography: high-resolution molecular imaging in pharmaceutical discovery and development.

Autoradiography (ARG) is a powerful, high resolution, quantitative molecular imaging technique used to study the tissue distribution of radiolabeled xenobiotics in biologic models. ARG involves the close apposition of solid specimens containing a radiolabeled substance to a detector layer, such as photographic emulsions, film, phosphor imaging plates and direct nuclear imagers/counters. The two basic types include: macroautoradiography, which is imaging of organs, organ systems and/or whole-body sections (WBA) and microautoradiography (MARG), which provides the localization of radioactivity at the cellular level. ARG has supported drug discovery and development efforts for many years and has provided pivotal decision making information for pharmaceutical research. This paper presents a review of the techniques, study designs and present considerations for use of WBA and MARG to support today's drug discovery and development efforts. In addition, this review comments on the integration of the ex vivo ARG and in vivo molecular imaging techniques to serve pharmaceutical discovery and development in the future.

Increased hepatobiliary clearance of unconjugated thyroxine determines DMP 904-induced alterations in thyroid hormone homeostasis in rats.

4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective antagonist of corticotropin releasing factor receptor-1 (CRF1 receptor) with an efficacious anxiolytic profile in preclinical animal models. In subchronic toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia, and a low incidence of follicular cell adenoma. The current investigations were designed to determine the mode of action by which DMP 904 disrupts thyroid homeostasis in male rats. Five-day treatment with DMP 904 (300 mg/kg/day) dramatically lowered serum thyroxine (T4) to levels below detectable limits (< 1 microg/dl) by 72 h, with concurrent decreases in triiodothyronine (T3, about a 70% decrease) and increases in thyroid stimulating hormone (TSH; about a three-fold increase). DMP 904 increased [125I]T4 total body clearance (Cl tb) (38.21 +/- 10.45 ml/h) compared to control (5.61 +/- 0.59 ml/h) and phenobarbital-treated rats (7.92 +/- 1.62 ml/h). This increase in Cl(tb) was associated with a significant increase in biliary clearance (Cl bile) of unconjugated [125I]T4 (nearly 80-times control rates) and increased liver:blood ratios of T4, suggestive of enhanced hepatic uptake of T4. A single dose of DMP 904 (200 mg/kg) increased mRNA levels of hepatic cytochrome P450s (CYP 3A1 and CYP 2B1) and UDP-glucuronosyltransferases (UGT 1A1 and UGT 1A2). DMP 904 also induced mRNAs of the canalicular transporter, multi-drug resistance protein-2 (Mrp2) and sinusoidal transporters, organic anion transporting proteins (Oatp1 and Oatp2) within 24 h. Western blot analysis confirmed DMP 904 related increases in Oatp2 protein expression. Collectively, these data suggest that DMP 904 is an agonist of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and that the decreased serum levels of T4 and T3 resulted from increased hepatobiliary clearance. However, DMP 904 is distinguished from other compounds associated with similar effects on thyroid hormone homeostasis because its effects were primarily related to increased biliary excretion of unconjugated T4.

Interaction of ritonavir on tissue distribution of a [(14)c]L-valinamide, a potent human immunodeficiency virus-1 protease inhibitor, in rats using quantitative whole-body autoradiography.

N-[(3-fluorophenyl)methyl]glycyl-N-[3-[((3-aminophenyl)sulfonyl)- 2-(aminophenyl)amino]-(1S,2S)-2-hydroxy-1-(phenylmethyl)propyl]- 3-methyl-L-valinamide (DPC 681, DPC(1)) on oral coadministration with ritonavir (RTV) in rats caused a significant increase in systemic exposure to DPC. Following a single oral dose of [(14)C]DPC with and without RTV pretreatment in rats, and subsequent analysis of whole-body sections, prepared at 1 and 7 or 8 h postdose, using whole-body autoradiography showed an increase in radioactivity in tissues (e.g., brain, and testes) upon coadministration. The distribution of radioactivity in the brain parenchyma and ventricles was different, such that the concentration of radioactivity was greater in cerebrospinal fluid (CSF) than in central nervous system. Thus, the use of CSF concentration of the total radioactivity as a surrogate for brain penetration would result in an overestimation. DPC was determined to be metabolized prominently by rCYP3A4. The increased tissue exposure to DPC in rats could largely be attributed to inhibition of CYP3A1/2 by RTV. DPC was also a good substrate for P-glycoprotein (Pgp), with K(m) of 4 microM and V(max) of 13 pmol/min. The Pgp-mediated transport of DPC across Caco-2 cells was readily saturated at >or=10 microM and was inhibited significantly by RTV at 5 to 10 microM. The data above and the reported RTV concentrations suggested that both the Pgp and CYP3A4 inhibition by RTV may play a significant role in enhancing the systemic and tissue exposure to DPC in humans.