A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29 -30 2-O-Me nucleotides, 1-2 other 2-modified nucleotides at positions U4 and U7, and a 3-3-linked nucleotide "cap." Eight 2-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2-NH 2 substitutions at U4 and U7, or 2-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only ϳ0.1 min. The addition of a 3-3-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000 -80,000-fold compared to the all-RNA parent ribozyme.Trans-acting ribozymes exert their activity in a highly specific manner and are therefore not expected to be detrimental to non-targeted cell functions. Because of this specificity, the concept of exploiting ribozymes for cleaving a specific target mRNA transcript is now emerging as a therapeutic strategy in human disease and agriculture (Cech, 1992;Bratty et al., 1993). For ribozymes to function as therapeutic agents, they may be introduced exogenously or produced endogenously in the target cells. In the former case, the chemically modified ribozyme must maintain its catalytic activity while also being stable to nucleases. A major advantage of chemically synthesized ribozymes is that site-specific modifications may be introduced at any position in the molecule. This approach provides flexibility in designing ribozymes that are catalytically active and stable to nucleases. In this manuscript we show that using this site-specific, chemical modification strategy, ribozymes can be designed that have wild-type catalytic activity and are not cleaved by nucleases.A variety of selective and uniform structural modifications have been applied to oligonucleotides to enhance nuclease resistance (Uhlmann and Peyman, 1990;Beaucage and Iyer, 1993;Milligan et al., 1993). Improvements in the chemical synthesis of RNA (Scaringe et al., 1990;Wincott et al., 1995) have led to the ability to similarly modify ribozymes containing the hammerhead ribozyme core motif Yang et al., 1992) (Fig. 1). Yang et al. (1992) demonstrated that 2Ј-O-Me modification of a ribozyme at all positions except G5, G8, A9, A15.1, and G15.2 (see numbering scheme in Fig. 1) led to a catalytically active molecule having a greatly decreased k cat value in vitro, but a 1000-fold increase in nuclease resistance over that of an all-RNA ribozyme when tested in a yeast extract. In another study (Paolella...

Purpose: African-American patients with colorectal cancer were observed to have increased 5-fluorouracil (5-FU)^associated toxicity (leukopenia and anemia) and decreased overall survival compared with Caucasian patients. One potential source for this disparity may be differences in 5-FU metabolism. Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme of 5-FU catabolism, has previously been shown to have significant interpatient variability in activity. Several studies have linked reduced DPD activity to the development of 5-FU toxicity. Although the distribution of DPD enzyme activity and the frequency of DPD deficiency have been well characterized in the Caucasian population, the distribution of DPD enzyme activity and the frequency of DPD deficiency in the African-American population are unknown. Experimental Design: Healthy African-American (n = 149) and Caucasian (n = 109) volunteers were evaluated for DPD deficiency using both the [2- 13C]uracil breath test and peripheral blood mononuclear cell DPD radioassay. Results: African-Americans showed significantly reduced peripheral blood mononuclear cell DPD enzyme activity compared with Caucasians (0.26 F 0.07 and 0.29 F 0.07 nmol/min/mg, respectively; P = 0.002). The prevalence of DPD deficiency was 3-fold higher in AfricanAmericans compared with Caucasians (8.0% and 2.8%, respectively; P = 0.07). African-American women showed the highest prevalence of DPD deficiency compared with African-American men, Caucasian women, and Caucasian men (12.3%, 4.0%, 3.5%, and 1.9%, respectively). Conclusion: These results indicate that African-Americans, particularly African-American women, have significantly reduced DPD enzyme activity compared with Caucasians, which may predispose this population to more 5-FU toxicity.5-Fluorouracil (5-FU) and its fluoropyrimidine derivatives (e.g., capecitabine) are widely prescribed in oncologic practice to treat gastrointestinal malignancies and are often used in the management of breast and head and neck cancer (1 -4). However, despite its widespread use, f31% of patients with advanced colorectal cancer who receive bolus 5-FU regimens experience grades 3 to 4 hematologic toxicities (5). The pharmacogenetic syndrome, dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) deficiency, has been shown to predispose cancer patients to severe 5-FU toxicity (6 -9). In particular, it is estimated that 40% to 60% of patients with cancer who present with severe 5-FU toxicity are 11).Several studies show the pivotal role of DPD in 5-FU metabolism and response. Earlier biochemical studies showed that DPD, the initial and rate-limiting enzyme of the pyrimidine catabolic pathway, degrades uracil, thymine, and 5-FU to dihydrouracil, dihydrothymine, and 5-fluoro-dihydrouracil, respectively (12, 13). Pharmacokinetic evaluation has further shown that DPD catabolizes >80% of an administered dose of 5-FU, thereby determining the amount of 5-FU available for anabolism (7). Furthermore, data from combined pharmacokinetic/pharmacodyna...

Purpose: Dihydropyrimidine dehydrogenase (DPD)-deficient cancer patients have been shown to develop severe toxicity after administration of 5-fluorouracil. Routine determination of DPD activity is limited by time-consuming and labor-intensive methods. The purpose of this study was to develop a simple and rapid 2-13 C-uracil breath test, which could be applied in most clinical settings to detect DPDdeficient cancer patients.Experimental Design: Fifty-eight individuals (50 "normal," 7 partially, and 1 profoundly DPD-deficient) ingested an aqueous solution of 2-13 C-uracil (6 mg/kg). 13 CO 2 levels were determined in exhaled breath at various time intervals up to 180 min using IR spectroscopy (UBiT-IR 300 ). DPD enzyme activity and DPYD genotype were determined by radioassay and denaturing high-performance liquid chromatography, respectively.Results: The mean (؎SE) C max , T max , ␦ over baseline values at 50 min (DOB 50 ) and cumulative percentage of 13 C dose recovered (PDR) for normal, partially, and profoundly DPD-deficient individuals were 186.4 ؎ 3.9, 117.1 ؎ 9.8, and 3.6 DOB; 52 ؎ 2, 100 ؎ 18.4, and 120 min; 174.1 ؎ 4.6, 89.6 ؎ 11.6, and 0.9 DOB 50 ; and 53.8 ؎ 1.0, 36.9 ؎ 2.4, and <1 PDR, respectively. The differences between the normal and DPD-deficient individuals were highly significant (all Ps <0.001).Conclusions: We demonstrated statistically significant differences in the 2-13 C-uracil breath test indices (C max , T max , DOB 50 , and PDR) among healthy and DPDdeficient individuals. These data suggest that a single time-point determination (50 min) could rapidly identify DPD-deficient individuals with a less costly and timeconsuming method that is applicable for most hospitals or physicians' offices.

We tested the hypothesis that the stable isotope [13 C]pantoprazole is O-demethylated by cytochrome P450 CYP2C19 and that the 13 CO 2 produced and exhaled in breath as a result can serve as a safe, rapid, and noninvasive phenotyping marker of CYP2C19 activity in vivo. Healthy volunteers who had been genotyped for the CYP2C19*2, CYP2C19*3, and CYP2C19*17 alleles were administered a single oral dose of [13 C]pantoprazole sodium-sesquihydrate (100 mg) with 2.1 g of sodium bicarbonate. Exhaled 13 CO 2 and 12 CO 2 were measured by IR spectroscopy before (baseline) and 2.5 to 120 min after dosing. Ratios of 13 CO 2 / 12 CO 2 after [ 13 C]pantoprazole relative to 13 CO 2 / 12 CO 2 at baseline were expressed as change over baseline (DOB). Maximal DOB, DOB 15 to DOB 120 , and area under the DOB versus time curve (AUC 0 -120 and AUC 0 -ϱ ) were significantly different among three genotype groups (CYP2C19*1/ *1, n ϭ 10; CYP2C19*1/*2 or CYP2C19*1/*3, n ϭ 10; and CYP2C19*2/*2, n ϭ 5) with predicted extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs) of CYP2C19, respectively (Kruskal-Wallis test, p Ͻ 0.01); linear regression analysis indicated a gene-dose effect relationship (r 2 ranged between 0.236 and 0.522; all p Ͻ 0.05). These breath test indices were significantly lower in PMs than IMs (p Ͻ 0.05) or EMs (p Ͻ 0.01) of CYP2C19. [13 C]Pantoprazole plasma exposure showed significant inverse correlation with breath test indices in the respective subjects (Pearson r ϭ Ϫ0.74; p ϭ 0.038). These feasibility data suggest that the [ 13 C]pantoprazole breath test is a reliable, rapid, and noninvasive probe of CYP2C19 and seems to be a useful tool to optimize drug therapy metabolized by CYP2C19.

Diagnostic (13)C-stable isotope probes are currently being expanded in their scope, to provide precise evaluations of the presence or absence of etiologically significant changes in metabolism due to a specific disease or the lack of a specific enzyme. The salient features of the (13)C-breath test are that they are non-invasive, non-radioactive, safe, simple, and effective. The simplicity of the (13)C-breath test makes it very applicable in a clinical setting: the physician can obtain valuable diagnostic information by distinguishing between two groups or populations on the basis of the recovery of (13)CO(2) from the ingested (13)C-substrate. The breath tests can also be used to monitor the progress of disease severity or to evaluate the efficacy of medications. This review concentrates on current research in the medical field dedicated to the metabolite (13)C-labelled carbon dioxide in exhaled air following ingestion of (13)C-labelled substrates.