Sudhir Agrawal

1 Basic Principles of Antisense Therapeutics.- 2 Antisense Medicinal Chemistry.- 3 In Vitro Cellular Uptake, Distribution, and Metabolism of Oligonucleotides.- 4 Pharmacokinetic Properties of Phosphorothioates in Animals - Absorption, Distribution, Metabolism and Elimination.- 5 Toxicity of Oligodeoxynucleotide Therapeutic Agents.- 6 Pharmacokinetic Properties of Phosphorothioate Oligonucleotides in Immune Stimulation by Oligonucleotides in Humans.- 7 Safety and Tolerance of Phosphorothioates in Humans.- 8 Inmune Stimulation by Oligonucleotides.- 9 Pharmacological Inhibition of Dopaminergic and Other Neurotransmitter Receptors Using Antisense Oligodeoxynucleotides.- 10 Pharmacological Effects of Antisense Oligonucleotide Inhibition of Immediate-Early Response Genes in the CNS.- 11 Inhibition of G Proteins by Antisense Drugs.- 12 Use of Antisense Oligonucleotides to Modify Inflammatory Processes.- 13 Antisense Oligonucleotides and Their Anticancer Activities.- 14 Pharmacological Activity of Antisense Oligonucleotides in Animal Models of Disease.- 15 Clincial Antiviral Activities.- 16 Antisense Oligonucleotides to Protein Kinase C-a and C-raf Kinase: Rationale and Clinical Experience in Patients with Solid Tumors.- 17 Nucleic Acid Therapeutics for Human Leukemia: Development and Early Clinical Experience with Oligodeoxynucleotides Directed at c-myb.- 18 Properties of ISIS 2302, an Inhibitor of Intercellular Adhesion Molecule-1, Humans.- 19 Pharmacokinetics and Bioavailability of Antisense Oligonucleotides Following Oral and Colorectal Administrations in Experimental Animals.- 20 Antisense Properties of Peptide Nucleic Acid.- 21 Triple Helix Strategies and Progress.

We describe preliminary studies of the pharmacokinetics, biodistribution, and excretion of an oligodeoxy-nucleotide phosphorothioate ([S]oligonucleotide) in mice. After either intravenous or intraperitoneal administration of a single dose (30 mg/kg of body weight), [S]oligonucleotide (35S-labeled at each internucleotide linkage) was found in most of the tissues for up to 48 hr. About 30% of the dose was excreted in urine within 24 hr, irrespective of the mode of administration; the excreted [S]oligonucleotide was found to be extensively degraded. In plasma, stomach, heart, and intestine, the [S]oligonucleotide was degraded by only 15%, whereas in the kidney and liver degradation was about 50% in 48 hr. The surprising observation was made that chain length extension of administered [S]oligonucleotide occurred in kidney, liver, and intestine. These results provide an initial definition of parameters for the pharmaceutical development of antisense oligonucleotides.

Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5 degrees C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 A. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of approximately 0.60 at 5 degrees C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was approximately 0.47 at 5 degrees C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.

Economic dispatch is a highly constrained optimization problem encompassing interaction among decision variables. Environmental concerns that arise due to the operation of fossil fuel fired electric generators, transforms the classical problem into multiobjective environmental/economic dispatch (EED). In this paper, a fuzzy clustering-based particle swarm (FCPSO) algorithm has been proposed to solve the highly constrained EED problem involving conflicting objectives. FCPSO uses an external repository to preserve nondominated particles found along the search process. The proposed fuzzy clustering technique, manages the size of the repository within limits without destroying the characteristics of the Pareto front. Niching mechanism has been incorporated to direct the particles towards lesser explored regions of the Pareto front. To avoid entrapment into local optima and enhance the exploratory capability of the particles, a self-adaptive mutation operator has been proposed. In addition, the algorithm incorporates a fuzzy-based feedback mechanism and iteratively uses the information to determine the compromise solution. The algorithm's performance has been examined over the standard IEEE 30 bus six-generator test system, whereby it generated a uniformly distributed Pareto front whose optimality has been authenticated by benchmarking against the epsiv -constraint method. Results also revealed that the proposed approach obtained high-quality solutions and was able to provide a satisfactory compromise solution in almost all the trials, thereby validating the efficacy and applicability of the proposed approach over the real-world multiobjective optimization problems.

Cleavage of the hepatitis C virus (HCV) polyprotein by the viral NS3 protease releases functional viral proteins essential for viral replication. Recent studies by Foy and coworkers strongly suggest that NS3-mediated cleavage of host factors may abrogate cellular response to alpha interferon (IFN-alpha) (E. Foy, K. Li, R. Sumpter, Jr., Y.-M. Loo, C. L. Johnson, C. Wang, P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr., Proc. Natl. Acad. Sci. USA 102:2986-2991, 2005, and E. Foy, K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr., Science 300:1145-1148, 2003). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of replicon-bearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.