Plant polyphenols and multidrug resistance: effects of dietary flavonoids on drug transporters in Caco-2 and MDCKII-MDR1 cell transport models.

The hypothesis tested was that specific flavonoids such as epicatechin gallate, epigallocatechin gallate, genistein, genistin, naringenin, naringin, quercetin and xanthohumol will modulate cellular uptake and permeability (P(e)) of multidrug-resistant substrates, cyclosporin A (CSA) and digoxin, across Caco-2 and MDCKII-MDR1 cell transport models. (3)H-CSA/(3)H-digoxin transport and uptake experiments were performed with and without co-exposure of the flavonoids. Aglycone flavonoids reduced the P(e) of CSA to a greater extent than glycosylated flavonoids with 30 microM xanthohumol producing the greatest effect (7.2 x 10(-6) to 6.6 x 10(-7) and 17.9 x 10(-6) to 4.02 x 10(-6) cm s(-1) in Caco-2 and MDCKII-MDR1 cells, respectively); while no measurable effects were seen with digoxin. Xanthohumol significantly demonstrated (1) saturable efflux, (2) increased uptake of (3)H-digoxin and (3) decreased uptake of (3)H-CSA in the Caco-2 cells. The transport data suggests that xanthohumol effects transport of CSA in a manner that is distinct from the digoxin efflux pathway and suggests that intestinal transport of these MDR1 substrates is more complex than previously reported.

Evidence of enhanced iron excretion during systemic phosphorothioate oligodeoxynucleotide treatment.

BACKGROUND: Phosphorothioate oligonucleotides, in general, possess properties that could be utilized in the development of therapeutic heavy metal chelators. METHODS: Iron excretion was measured in 16 patients participating in studies to test the safety of OL(1)p53, a 20-mer phosphorothioate oligonucleotide complementary to p53 mRNA. Patients were given OL(1)p53 at doses of 0.05 to 0.25 mg/kg/h for 10 days by continuous intravenous infusion. Urine was collected during the study and analyzed for iron, copper, cadmium, and zinc. RESULTS: We found that phosphorothioate oligonucleotides have a high affinity for iron as well as several other clinically relevant toxic metals. Analysis of patient urine following administration of OL(1)p53 reveals a 7.5-fold increase in iron excretion at low doses (0.05 mg/kg/h). CONCLUSIONS: Phosphorothioate oligonucleotides may have therapeutic potential as heavy metal chelators. Low doses of phosphorothioate oligonucleotide facilitated the excretion of iron. Renal clearance of iron-phosphorothioate oligonucleotide complexes most likely involves secretion into proximal tubules.

Pharmacokinetics and in vivo effects of a six-base phosphorothioate oligodeoxynucleotide with anticancer and hematopoietic activities in swine.

A short phosphorothioate oligodeoxynucleotide telomere mimic with the sequence 5'-d(TTAGGG)-3', TAG-6, has been shown to inhibit telomerase activity and have antineoplastic and hematopoietic stimulatory properties. In this study, three immature male domestic swine (weighing approximately 40 kg) were administered 200 mg/m2 of TAG-6 by continuous intravascular infusion at rates of 0.48 +/- 0.07 mg/hr for 14 days to evaluate the pharmacokinetics, toxicity, and tissue distribution. There was considerable variability (both within each animal and across animals) observed in the pharmacokinetic data. The plasma half-life (t1/2 appeared to be short enough that it could be assumed that steady state was attained by at least 96 h after the start of the infusion. The t1/2 estimates for the three pigs were 8.96, 109, and 1.97 h (the long t1/2 for pig 2 may be explained by poor parameter estimation due to the variability). The volume of distribution ranged from 9.80 to 51.8 L (0.3-1.4 L/kg), and plasma clearance estimates ranged from 0.33 to 3.46 L/h (5.5-57.7 ml/min). The average plasma concentrations at steady state were 0.845, 0.933, and 0.178 microg/ml (0.44, 0.49, and 0.093 microM) for the three animals. Nearly 30% of the administered dose was cleared through renal excretion by day 7 postinfusion. The distribution of TAG-6 was primarily to the liver and kidney, but the spleen and thyroid accumulated relatively high concentrations of TAG-6. TAG-6 was metabolized to apparently higher molecular weight products, which were observed in the urine. The size periodicity of these apparently higher molecular weight products was in 6-base intervals, which is consistent with the actions of telomerase. The infusion did not produce significant changes in serum chemistry or circulating blood cells, but a decrease in colony-forming unit-granulocyte-monocyte (CFU-GM) colony formation from BM was observed. These data suggest that TAG-6 may be a very specific pharmacophore.

Bolus intravenous injection of phosphorothioate oligonucleotides causes hypotension by acting as alpha(1)-adrenergic receptor antagonists.

Bolus intravenous injections of phosphorothioate oligonucleotides (PS-ODN) into primates cause profound hypotension, which has been attributed to complement activation, the biochemical pathway leading to acute inflammatory response. Because the hypotension was not accompanied by peripheral or pulmonary edema and epinephrine was not effective, but administration of 200 ml Ringer's lactate was effective, we examined the possibility that the 15-base PS-ODN interferes with sympathetic tone. We administered doses ranging from 3.3 to 10 mg/kg of a 15-base PS-ODN as a 30-60 s iv bolus into the right atrium of conscious Macaca mulatta. Blood pressure fell to 27 mm Hg following a 5.0 mg/kg dose, but no hypotension was observed after a 3.3 mg/kg dose; 10 mg/kg was lethal. Adrenergic receptor binding was evaluated in radioligand binding assays using rat cerebral cortex membranes with radiolabeled prazosin. The 15-base PS-ODN competes with prazosin for the alpha(1)-adrenergic receptor with an IC50 of 14 microM, which favors binding over serum albumin (K(d) = 37 to 48 microM). Admixing serum albumin with 5.0 mg/kg 15-base PS-ODN prior to injection prevented hypotension, suggesting that unbound PS-ODN interferes with sympathetic tone before binding to plasma proteins. Interactions of the 15-base PS-ODN with the alpha(1)-adrenergic receptor in vivo were confirmed by a decreased response to phenylephrine. Reducing the length from 15 to 9 or 5 bases abolished alpha(1)-adrenergic receptor binding in vitro and bolus infusion of 5.0 mg/kg of 9-base PS-ODN no longer produced hypotension. In conclusion, the 15-base PS-ODN shows cooperative binding to the alpha(1)-adrenergic receptor, which produces cardiovascular effects that are oligomer length, dose, and formulation dependent.

The cytotoxic effects of single-stranded telomere mimics on OMA-BL1 cells.

Telomerase is a ribonucleoprotein that adds 5'-d(TTAGGG)-3' hexameric repeats onto the 3' ends of chromosomes. High telomerase activity has been associated with immortal cells, transformed cells, mitogenic stimulation, and proliferative diseases. It is not clear what phenotype would be observed by transient inhibition of telomerase. Studies were designed to inhibit telomerase activity using a series of S-ODN telomere sequence motifs. The studies evaluated the length, hydrogen bonding, and sequence requirements of telomerase inhibition using the TRAP assay and a bioassay measuring cell viability following exposure to the compounds. In addition, we have also studied the role of the 3' end and secondary structure of telomere mimics on telomerase inhibition. Observations reveal that sensitivity to the S-ODNs may not require hybridization to an antisense target but required guanine nucleotides on the 3' end for cells in culture and telomerase inhibition in vitro. The importance of H bonding and the requirement for a free 3' end for the activity of these compounds has also been demonstrated. However, transient inhibition of telomerase is not cytotoxic to all immortal cells and is not sufficient to explain the mechanism of cytotoxicity of these short oligonucleotides.

Tumor cell growth is inhibited by suppressing metallothionein-I synthesis.

The effect of metallothionein (MT)-I antisense oligodeoxynucleotide (ODN) on the growth of three kinds of tumor cells was studied, since MTs may be involved in cell growth. When MT-I antisense ODN was added to leukemia P388 cells, cell growth was inhibited in a manner dependent on the dose and incubation time. MT-I antisense ODN was also inhibitory for other tumor cell lines, i.e. Ehrlich carcinoma and sarcoma 180. A significant decrease in the level of MT, but not of Zn, was observed in MT-I antisense ODN-treated cells. On the other hand, control ODN did not inhibit the cell growth appreciably. These results indicate that MT-I expression may be necessary for the growth and survival of these tumor cells.

A hexameric phosphorothioate oligonucleotide telomerase inhibitor arrests growth of Burkitt's lymphoma cells in vitro and in vivo.

A phosphorothioate oligonucleotide (PS-ODN) with sequence identical to the repeat sequence of the mammalian telomere, 5'-d(TTAGGG)-3', was incubated with a Burkitt's lymphoma-derived (OMA-BL1) cell line. This hexanucleotide inhibits telomerase activity in cell lysates, lengthens cell doubling time, and induces apoptosis. Concatenated repeats (12-, 18-, and 24-mers) of the 5'-d(TTAGGG)-3' motif induce similar cellular responses. Scrambled sequences do not efficiently inhibit telomerase activity or significantly alter cell growth and viability. The in vivo efficacy of this PS-ODN was evaluated in a xenograft human-nude mouse model. Once tumors were established these animals were administered the telomere mimic, 5'-d(TTAGGG)-3', a control scrambled sequence 5'-d(TGTGAG)-3', or saline for 14 days via a subcutaneous osmotic pumps in a blinded study monitoring tumor size with dose and time. A significant decrease in tumor size was observed in animals given 50 micrograms/mouse/day 5'-d(TTAGGG)-3', but not following 5'-d(TGTGAG)-3', relative to the saline-treated animals. The antitumor activity of the 6-mer telomere mimic demonstrated a dose dependency including a reduction in metastatic nodules in the spleen. No activity was observed with the scrambled controls. In addition to antitumor activity we observed an increase in the mouse hematopoietic progenitor cell populations, BFU-e and CFU-GM. These results demonstrated the effects of a short hexameric oligonucleotide telomere mimic in vitro and in vivo and the potential utility of short oligonucleotides as telomerase inhibitors.

Synthetic antisense oligonucleotide probes the essentiality of metallothionein gene.

The metallothionein II gene, whose structure is highly conserved throughout the animal kingdom, is composed of three exons and two introns. Synthetic antisense oligonucleotides (ODN), with sequence complementary to the messenger RNA coding for human metallothionein II, were prepared and tested for their ability to inhibit both constitutive- and cadmium-induced metallothionein protein synthesis in human Chang liver cells and hamster lung V79 cells in culture. The property of sense ODN was also examined as a measure of sequence specificity. The antisense inhibition of metallothionein protein synthesis rendered all cell lines more susceptible to the toxic effects of cadmium. However, the sense ODN had no effects on either metallothionein protein synthesis or sensitivity to cadmium. Phosphorothioate oligonucleotides were more potent in enhancing the toxicity of cadmium than phosphodiester oligonucleotides. The antisense oligonucleotides targeted to the splice donor region between exon 1 and intron 1 was significantly more effective as an inhibitor than those targeted to either the 5' end of the mRNA or within exon 3. Therefore, not all antisense oligonucleotide sequences are equally efficient as inhibitors of metallothionein synthesis. Therefore, the delineation of an obvious mechanism for predicting the most potent sequence is not apparent at this time. The results of this study are interpreted to indicate that (a) metallothionein is endowed with an essential gene, (b) modifications in oligonucleotide structures show specificity in inhibiting metallothionein synthesis, and (c) antisense inhibition of metallothionein gene expression may provide a useful tool in studying the action and perhaps the function(s) of metallothionein.