**1. Introduction**

678 Non-Viral Gene Therapy

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G.R., Smith, T.D., Rauch, C., and Smith, C.A. (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. Pyrrole(Py)-imidazole(Im)(PI) polyamides are small synthetic molecules composed of aromatic rings of *N*-methylpyrrole and *N*-methylimidazole amino acids (Trauger et al., 1996). Synthetic polyamides recognize and bind to specific nucleotide sequences in the minor groove of double-helical DNA with high affinity (Pilch et al., 1996). Various sequencespecific DNA-binding PI polyamides have been developed to regulate gene expression by targeting the promoter regions of enhancer and transcription factor-binding elements in vitro (Murty et al., 2004). PI polyamides were first identified from duocarmycin A and distamycin A, which bind in the minor groove of DNA (Tao et al., 1999; Trauger et al., 1996). Sequence-specific DNA recognition by PI polyamide depends on the sequence of side-byside amino acid pairs. A pair of Py opposite Im targets the CG base pair, whereas Im opposite Py recognizes the GC base pair, and the Py/Py combination binds to both AT and TA base pairs (White et al., 1997). PI polyamides designed to bind to the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and the TATAbox-binding protein DNA binding site have been shown to inhibit virus replication in isolated human peripheral blood lymphocytes (Dickinson et al., 1998).

PI polyamides can easily enter into the nucleus and bind to chromosomal DNA. Many promising observation for gene therapy using PI polyamides have been reported. PI polyamide targeting rat transforming growth factor (TGF)-β1 has been reported to inhibit the expressions of TGF-β1 mRNA and protein in the renal cortex of Dahl-S rats. The targeted PI polyamide also reduced glomerulosclerosis and interstitial fibrosis without side effects. These observations indicate that PI polyamides will be effective for TGF-β1-related diseases, including progressive renal injury (Matsuda et al., 2011; Matsuda et al., 2006). PI polyamides targeting human aurora kinase-A (AURKA) and -B (AURKB) promoters significantly inhibited the promoter activities, and mRNA and protein expression levels of AURKA and

Pyrrole-Imidazole Polyamides for Gene Therapy: Bioanalytical Methods and Pharmacokinetics 681

High-performance liquid chromatography (HPLC) has been used for many years as a useful and conventional tool for the analysis of a drug. Bioanalytical methods by HPLC with UV detection were developed for the determination of PI polyamides A and B in the rat matrix. Sample extraction is one of the important steps and key to success in constructing a robust method. A simple protein precipitation method was developed for the extraction of PI polyamides A and B from rat plasma, whereas solid phase extraction was carried out to extract PI polyamides A and B from rat urine and bile, because a large number of urinary and biliary matrices can interfere with the compounds. It is important to determine the rates of urinary and biliary excretions because these excretions play pivotal roles in the elimination pathway of a drug. The developed methods were successively validated for selectivity, sensitivity, linearity, accuracy, and precision, following the guideline for Bioanalytical Method Validation published by Food and Drug Administration in 2001. Chromatographic separation was conducted using a reversed-phase TSK-GEL ODS-80TM (4.6 mm x 150 mm) column maintained at 40 ºC. The mobile phase of solvent A was 0.1% acetic acid and that of solvent B was acetonitrile (a linear increase from 0 to 80% B over 10 min (plasma and urine) or 35 min (bile) and an isocratic flow at 60% B for 5 min). The flow rate was set at 1.0 mL/min (plasma and urine) or 0.75 mL/min (bile). The detection wavelength was set at 310 nm. PI polyamides A and B were well separated from the coextracted material under the described chromatographic conditions at approximate retention times of 9.7 (25.0 in bile) and 10.5 min, respectively. The peak shapes were satisfactory and completely resolved from one another. No interference from rat matrices

Fig. 2. Representative chromatograms of blank rat plasma (A), blank rat urine (B) and blank rat bile (C) spiked with PI polyamide A, and blank rat plasma (D), blank rat urine (E) and blank rat bile (F) spiked with PI polyamide B. The concentrations of PI polyamides were 5

Table 1 shows the intra- and inter-assay precision and accuracy of PI polyamides A and B. The intra- and inter-assay accuracies (RE) were within ± 20% for the lower limit of

**2. Bioanalytics** 

was observed (Fukasawa et al., 2007).

(A), 20 (B), 1 (C), 5 (D), 20 (E) and 5 (F) µg/mL.

AURKB. They also demonstrated a marked antiproliferative synergy in human tumor cell lines as a result of induction of apoptosis-mediated severe catastrophe of cell-cycle progression (Takahashi et al., 2008). PI polyamides specifically inhibited lectin-like oxidized low-density lipoprotein receptor-1 mRNA expression and apoptosis induced by oxidized low-density lipoprotein and angiotensin II in human umbilical vein endothelial cells (Ueno et al., 2009). From these observations, PI polyamides have been identified as novel candidates for gene therapy.

Pharmacokinetics is the science that studies the behavior of a circulating drug administered to a body, mainly focusing on absorption, distribution, metabolism, and excretion (ADME) of a drug (Jang et al., 2001). The concentration of a drug in a body can be obtained by a bioanalytical method which includes sample extraction and detection of a drug, and the obtained data are analyzed to evaluate the pharmacokinetics of the drug. Needless to say, a robust bioanalytical procedure is crucial for evaluating the appropriate pharmacokinetic profile of a drug.

In this chapter, we show the bioanalytical procedure, pharmacokinetics, and modeling of PI polyamides A and B. PI polyamides A and B are illustrated in Fig. 1. PI polyamide A was composed of Ac-ImPyPy-ImPyPy-β-Dp (β, β-alanine; Dp, *N*, *N*-dimethylaminopropylamide). PI polyamide B was composed of Ac-PyPy-β-PyImPy-PyPyPy-β-ImPy-β-Dp. The molecular weights of PI polyamides A and B were calculated from the sum of the standard atomic weights of all the atoms (Wieser, 2006). The molecular weights of PI polyamides A and B are 1035.12 and 1665.78, respectively. PI polyamide B was designed to bind to the activator protein-1 (AP-1)-binding site of the TGF-β1 promoter, whereas PI polyamide A also, with a hairpin structure, was designed for comparing with other types of PI polyamide with a hairpin structure and a higher molecular weight.

Fig. 1. Chemical structures of PI polyamides A and B.
