Propofol hemisuccinate is a prodrug water soluble form of the lipophilic, phenolic compound propofol (2,6-di-isopropylphenol), that is the active ingredient in the widely used anesthetic agent Diprovan. Propofol binds to GABAA receptors but also has a phenolic structure that confers antioxidant properties to the molecule. The effects of propofol hemisuccinate in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses and time regimes. Propofol hemisuccinate, 100 mg/kg given three times a day from day 7 or day 12 until day 16 after disease initiation, significantly reduced maximal EAE score. Histology studies supported the clinical findings demonstrating reduction in the inflammatory response in the lumbar spinal cord in animals treated with propofol hemisuccinate. Decreased levels of nitrotyrosine and unchanged levels of induced nitric oxide synthase suggest propofol hemisuccinate crossed the blood brain barrier and exerted its effects by lowering reactive oxygen species levels. The results suggest that propofol hemisuccinate may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis.

“Hydrolytic metalloenzymes are obiquitous in nature and many models have been studied. Detailed mechanistic analyses on simple enzyme models can give information on how enzymes work. This review article focusses on artificial hydrolytic metalloenzymes and examines a mechanistically unified approach to the hydrolysis of esters, amides, nitriles and phosphate esters.”

Nonviral gene delivery has great potential for replacement of recombinant protein therapy. In many cases, gene therapies would be a considerable improvement over existing therapies because of putative advantages in dosing schedule, patient compliance, toxicity, immunogenicity, and cost. Development of a nonviral gene delivery vehicle capable of efficient, cell-specific delivery will be a valuable addition to the clinical armamentarium.KeywordsNuclear Magnetic Resonance SpectroscopyCationic PolymerCharge RatioLithium HydroxidePhoenix PharmaceuticalThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

After a single IV injection of the water-soluble propofol prodrug propofol phosphate (PP) in mice, rats, rabbits, and pigs, propofol was produced rapidly (1-15 min), inducing dose-dependent sedative effects. In mice, the hypnotic dose (HD50), lethal dose (LD50), and safety index (defined as a ratio: LD50/HD50) were 165.4 mg/kg, 600.6 mg/kg, and 3.6, respectively. Propofol was produced with half-lives of 5.3 +/- 0.6 min in rats, 2.1 +/- 0.6 min in rabbits, and 4.4 +/- 2.4 min in pigs. The maximal concentration was dose and species dependent. The elimination half-life was 24 12 min in rats, 21 16 min in rabbits, and 225 +/- 56 min in pigs. Propofol generated from PP produced pharmacological effects similar to those described in the literature. We found a correlation between PP dose and duration of sedation with propofol concentrations larger than 1.0 mug/mL, which produced somnolence and sedation in rats and pigs. Adequate sedation and, at large enough doses, anesthetic-level sedation were produced after the administration of PP. Overall, PP, the water-soluble prodrug of propofol, seems to be a viable development candidate for sedative and anesthetic applications.

Chronic viral hepatitis is a major clinical problem, with over half a billion persons infected worldwide. Current therapies, principally treatment with recombinant IFN-alpha protein, have limited benefit. Recent studies suggest that gene-based expression of IFN-alpha is a possible therapeutic alternative that may improve the effectiveness of treatment. Gene delivery to the liver and consequent IFN-alpha expression therein, has the potential to concentrate the protein at the target organ and provide more continuous exposure to the therapeutic agent. Other potential gene and nucleic acid therapeutics for viral hepatitis are also being investigated. Key to the deployment of these future therapies is a suitable method of gene delivery. Although recombinant viral vector systems, such as adenovirus, are currently the most effective means of gene delivery to the liver, their use presents many concerns. These include immune and inflammatory reactions to the viral vector and possible adverse interactions between the recombinant virus and the pre-existing viral infection. Non-viral gene delivery systems would be a preferred treatment modality. The efficiency of current non-viral systems is not adequate for systemically administered liver gene therapy. However, recent use of membrane permeabilisation techniques has shown that high efficiency non-viral gene transfer agents are possible. The future coupling of these improved delivery systems with gene- or nucleic acid-based therapeutics currently in development holds out great promise for new generations of antihepatitis therapies.

This review focuses on recent progress and novel strategies to improve the efficiency of in vivo non-viral gene delivery. Examples of the most promising attempts to overcome specific barriers are presented in fuller detail. Current research into several of the most difficult steps in the gene delivery pathway is discussed including particle stabilization, targeting, cytoplasmic entry and access to the nucleus. The impact of recent reports on our current understanding of the true limitations to in vivo delivery is also discussed. The importance of preclinical animal models for the development of clinical applications of gene therapy is noted.

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.

Polycations have been used for gene delivery in vitro quite successfully, however, in vivo applications suffered from serum effects that lower the overall gene drug efficiency. PEG polymers have been used extensively to minimize serum effects and create "stealth liposomes", biocompatible materials, and proteins with extended circulation. Here, we report our efforts towards creating "stealth polyplexes". A comb-type polycation, poly-L-lysine-graft-PEG copolymers were successfully prepared by ring opening of PEG-epoxide with E-amino lysine groups of linear poly-L-lysine. The ratios of PEG-epoxide to poly-L-lysine, PEG-epoxide size (2K, 3K, and 5B), and poly-L-lysine size (10K, 26K, and 38K) were varied. Copolymers with as little as 2% grafted PEG chains sterically stabilized DNA/copolymer complexes (polyplexes) even at charge neutrality. These polyplexes, formed with copolymers with various size of PEG chains grafted on various lenghths of poly-L-lysine backbone, remained relatively small, approximately 100 nm in saline With higher degree of grafting, the binding was significantly diminished. In addition, the morphology of polyplexes changed from thoroidal to more elongated, worm-like forms. Some globular structures were detected in cases of a lower degree of grafting. Finally, DNA release form polyplexes when exposed to negatively charged macromolecules, poly-L-aspartic acid sodium salt, is very structure dependant. Enhanced levels of luciferase expression observed with PLL-PEC polyplexes versus either free DNA or PLL polyplexes are encouraging and warrant further optimization of the polymeric gene delivery system.

The equilibrium constant for chelation of carboxylates to cis-diaqua tetraamine Co(III) complexes is highly sensitive to the tetraamine ligand structure, basicity of the chelating carboxylate, and solvent polarity. cis-Diaqua tetraamine Co(III) complexes that easily accommodate chelation of carboxylates are also highly reactive for hydrolyzing carboxyl esters, amides, nitriles, and phosphate esters. The X-ray crystal strcuture of [(trpn)Co(eta(2)-O2CC(CH3)(3)]-(ClO4)(2) has been determined: trpn = tris(3-aminopropyl)amine, C14H33N4O10Cl2CO, orthorhombic, P2(1)2(1)2(1), a = 8.7822(3)Angstrom, b = 14.9959(6)Angstrom, c = 17.5277(5)Angstrom, V = 2308.35(14) Angstrom(3), Z = 4.

Carboxyl groups are important for efficient renal uptake of small anionic molecules. [(TCO)-T-99m(ECH)](2-) (ECH = pentaanionic form of (2R,7R)-2,7-dicarboxy-3,6-diaza-1,8-octanedithiol (ECH(6))) is a potentially useful radiopharmaceutical for diagnosing renal function. As typically isolated, the [(TC)-T-99(V)O](3+) and [Re(V)O](3+) derivatives of ECH(6) are neutral trifunctional acids, e.g., (TCO)-T-99(ECH(3)) and ReO(ECH(3)). The ligand has two carboxyl groups; the presence of the oxo group on the metal lowers the symmetry of the coordinated ligand, and one carboxyl group is syn and the other anti to the oxo group. Extremely broad NMR spectra at physiological pH and difficulty in crystallization have precluded complete characterization. Both the neutral species, ReO(ECH(3)) (3), and the ammonium salt of the trianionic species, [NH4](3)[ReO(EC)].C4H8O2 (5), have now been structurally characterized by X-ray diffraction. The coordination geometry of 3 is distorted octahedral, with the notable and unexpected feature that the anti-carboxylate group is coordinated trans to the oxo ligand. The coordination geometry of 5 is distorted square pyramidal, with the unusual feature that both nitrogen donor atoms are deprotonated. To determine the causes for the very broad H-1 and C-13 spectra of the Tc and Re complexes in aqueous solutions near physiological pH, we first prepared the analogous N2S2 ligand (TMECH(6)) incorporating penicillamine (pen), which has no CH coupling. (The ECH(6) ligand is derived from cysteine (cys), which has a coupled three-spin system.) The acid dissociation constants of the Re EC and TMEC complexes were found to be similar, with one NH group partially deprotonated under physiological conditions. We then applied a battery of 2D NMR methods to Re derivatives of both N2S2 ligands in D2O. From these studies, the EC H-1 NMR spin systems were identified and assigned for both acidic and basic aqueous solutions. From the Karplus relationship, the relevant torsion angles were calculated and compared to those determined crystallographically. These studies demonstrated that the anti-carboxyl group was coordinated in aqueous solution at low pH but not at high pH. The NMR results for ReO(TMECH(3)) under physiological conditions are consistent with two forms: (i) and anti-cys NH deprotonated, anti-carboxyl dissociated form and (ii) an anti-cys NH, coordinated anti-carboxyl form. The unusual broadening of ReO(ECH(3)) and TcO(ECH(3)) signals at pH 7 can be very confidently attributed to the coupling of carboxyl coordination and NH proton dissociation. Thus, proton dissociation makes the N a better donor. The resulting electronic and geometric changes at N favor deligation of the carboxyl group. On the NMR time scale, this process is in the intermediate exchange domain and leads to broad lines. The apparent pK(a) near physiological pH is net a simple pK(a) the N2S2 ligand has different denticity in the acid and conjugate base forms of the complex. Molecular mechanics modeling suggests that anti-cys NH deprotonation leads to increased strain on the six-coordinate form, whereas syn-cys NH deprotonation has little effect. The most strain-free NH deprotonated quadridentate form has the anti-cys NH deprotonated. Thus, the calculations and the results of solution NMR and solid-state crystallography are all consistent.

Hydroxyethyl phosphate forms a monodentate complex with [(tren)Co(OH2)2]3+ whereas it forms a bidentate complex with [(trpn)Co(OH2)2]3+ (tren = tris(2-aminoethyl)amine; trpn = tris(3-aminopropyl)amine). The monodentate phosphate hydrolyzes more rapidly (k = 2 X 10(-5) s-1 at pH 6.0, 60-degrees-C) than the bidentate phosphate. Although the chelated phosphate is highly resistant to hydrolysis, it is rapidly hydrolyzed by additional [(trpn)Co(OH2)2]3+ (k = 1 X 10(-2) M-1 s-1 at pH 6.0, 25-degrees-C).

NMR data demonstrate that the negative charge of 2,3,4,5,6-pentamethylbenzyl anion is confined to a single resonance-stabilized methylene. No evidence was found for a fluxional carbanion in which protons pass rapidly, in a circular fashion, from one carbon to the next.