Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration

'Stealth' liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 micromol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 micromol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.     

Reconstitution mechanism of nucleosome core particles mediated by poly(l-glutamic acid)

Poly(l-glutamic acid) has been reported to mediate in vitro nucleosome assembly (Stein, A., Whitlock, J.P., Jr. and Bina, M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5000–5004). To study the reaction mechanism, we have reconstituted nucleosome core particles from chicken erythrocyte core DNA and core histones in the presence of poly(l-glutamic acid) and analyzed the assembly products by polyacrylamide gel electrophoresis. Poly(l-glutamic acid), which binds and forms a large complex with core histones, is replaced with core DNA in the reconstitution process. When histone-poly(l-glutamic acid) complex and core DNA are mixed with a histone:DNA ratio of 1.0, the yield of core particles increases by prolonged reconstitution time. Two phases with a distinct time range appear in the process. In the fast phase within 30 min, 60% of the DNA is involved in products containing histones: reconstituted core particles, a larger nucleoprotein complex and aggregation. In the second phase, the remaining DNA and the DNA in the aggregation decrease, and the core particles increase slowly. The yield of core particles is approx. 60% after 24 h. The slow phase is not observed by reconstitution with a histone:DNA ratio of 2.0 in the initial mixture. The reaction scheme of the assembly process derived from these data is given. Based on the in vitro reaction scheme, the possible role of in vivo ‘nucleosome assembly factors’ is also discussed.     

Effects of acute and sub-chronic l-dopa therapy on striatal l-dopa methylation and dopamine oxidation in an MPTP mouse model of Parkinsons disease

Aims

The molecular mechanisms for the loss of 3,4-dihydroxyphenylalanine (l-dopa) efficacy during the treatment of Parkinson's disease (PD) are unknown. Modifications related to catecholamine metabolism such as changes in l-dopa and dopamine (DA) metabolism, the modulation of catecholamine enzymes and the production of interfering metabolites are the primary concerns of this study.

Main methods

Normal (saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pre-treated mice were primed with 100 mg/kg of l-dopa twice a day for 14 days, and a matching group remained l-dopa naïve. l-dopa naive and primed mice received a challenge dose of 100 mg/kg of l-dopa and were sacrificed 30 min later. Striatal catecholamine levels and the expression and activity of catechol-O-methyltransferase (COMT) were determined.

Key findings

Normal and MPTP pre-treated animals metabolize l-dopa and DA similarly during l-dopa therapy. Administration of a challenge dose of l-dopa increased l-dopa and DA metabolism in l-dopa naïve animals, and this effect was enhanced in l-dopa primed mice. The levels of 3-OMD in MPTP pre-treated animals were almost identical to those in normal mice, which we found are likely due to increased COMT activity in MPTP pre-treated mice.

Significance

The results of this comparative study provide evidence that sub-chronic administration of l-dopa decreases the ability of the striatum to accumulate l-dopa and DA, due to increased metabolism via methylation and oxidation. This data supports evidence for the metabolic adaptation of the catecholamine pathway during long-term treatment with l-dopa, which may explain the causes for the loss of l-dopa efficacy.     

Polyelectrolyte nanoparticles based on water-soluble chitosan-poly(l-aspartic acid)-polyethylene glycol for controlled protein release

Water-soluble chitosan (WSC)-poly(l-aspartic acid) (PASP)-polyethylene glycol (PEG) nanoparticles (CPP nanoparticles) were prepared spontaneously under quite mild conditions by polyelectrolyte complexation. These nanoparticles were well dispersed and stable in aqueous solution, and their physicochemical properties were characterized by turbidity, FTIR spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), and zeta potential. PEG was chosen to modify WSC-PASP nanoparticles to make a protein-protective agent. Investigation on the encapsulation efficiency and loading capacity of the bovine serum albumin (BSA)-loaded CPP nanoparticles was also conducted. Encapsulation efficiency was obviously decreased with the increase of initial BSA concentration. Furthermore, its in vitro release characteristics were evaluated at pH 1.2, 2.5, and 7.4. In vitro release showed that these nanoparticles provided an initial burst release, followed by a slowly sustained release for more than 24 h. The BSA released from CPP nanoparticles showed no significant conformational change compared with native BSA, which is superior to the BSA released from nanoparticles without PEG. A cell viability study suggested that the nanoparticles had good biocompatibility. This nanoparticle system was considered promising as an advanced drug delivery system for the peptide and protein drug delivery.     

Molecular aspects of the bilayer stabilization induced by poly(l-lysines) of varying size in cardiolipin liposomes

The interaction between poly(l-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and ³¹P- and ¹³C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca²⁺ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca²⁺, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca²⁺ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca²⁺, the lipid was organized in the hexagonal H_II phase in the presence of Ca²⁺. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes.     

Stereosensitive formation and interconversion of sulfur-bridged cobalt(III)-mercury(II) polynuclear complexes with d- and/or l-penicillaminates

The reaction of trans(N)-[Co(d-pen)₂]⁻ (pen = penicillaminate) with HgCl₂ or HgBr₂ in the molar ratios of 1:1 gave the sulfur-bridged heterodinuclear complex, [HgX(OH₂){Co(d-pen)₂}] (X = Cl (1a) or Br (1b)). A similar reaction in the ratio of 2:1 produced the trinuclear complex, [Hg{Co(d-pen)₂}₂] (1c). The enantiomers of 1a and 1c, [HgCl(OH₂){Co(l-pen)₂}] (1a′) and [Hg{Co(l-pen)₂}₂] (1c′), were also obtained by using trans(N)-[Co(l-pen)₂]⁻ instead of trans(N)-[Co(d-pen)₂]⁻. Further, the reaction of cis · cis · cis-[Co(d-pen)(l-pen)]⁻ with HgCl₂ in the molar ratio of 1:1 resulted in the formation of [HgCl(OH₂){Co(d-pen)(l-pen)}] (2a). During the formations of the above six complexes, 1a, 1b, 1c, 1a′, 1c′, and 2a, the octahedral Co(III) units retain their configurations. On the other hand, the reaction of cis · cis · cis-[Co(d-pen)(l-pen)]⁻ with HgCl₂ in the molar ratio of 2:1 gave not [Hg{Co(d-pen)(l-pen}₂] but [Hg{Co(d-pen)₂}{Co(l-pen)₂}] (2c), accompanied by the ligand-exchange on the terminal Co(III) units. The X-ray crystal structural analyses show that the central Hg(II) atom in 1c takes a considerably distorted tetrahedral geometry, whereas that in 2c is of an ideal tetrahedron. The interconversion between the complexes is also examined. The electronic absorption, CD, and NMR spectral behavior of the complexes is discussed in relation to the crystal structures of 1c and 2c.     

Reconstitution mechanism of nucleosome core particles mediated by poly(l-glutamic acid)

Poly(l-glutamic acid) has been reported to mediate in vitro nucleosome assembly (Stein, A., Whitlock, J.P., Jr. and Bina, M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5000–5004). To study the reaction mechanism, we have reconstituted nucleosome core particles from chicken erythrocyte core DNA and core histones in the presence of poly(l-glutamic acid) and analyzed the assembly products by polyacrylamide gel electrophoresis. Poly(l-glutamic acid), which binds and forms a large complex with core histones, is replaced with core DNA in the reconstitution process. When histone-poly(l-glutamic acid) complex and core DNA are mixed with a histone:DNA ratio of 1.0, the yield of core particles increases by prolonged reconstitution time. Two phases with a distinct time range appear in the process. In the fast phase within 30 min, 60% of the DNA is involved in products containing histones: reconstituted core particles, a larger nucleoprotein complex and aggregation. In the second phase, the remaining DNA and the DNA in the aggregation decrease, and the core particles increase slowly. The yield of core particles is approx. 60% after 24 h. The slow phase is not observed by reconstitution with a histone:DNA ratio of 2.0 in the initial mixture. The reaction scheme of the assembly process derived from these data is given. Based on the in vitro reaction scheme, the possible role of in vivo ‘nucleosome assembly factors’ is also discussed.     

Fabrication of chronocoulometric DNA sensor based on gold nanoparticles/poly(l-lysine) modified glassy carbon electrode

In this work, we fabricated a sensitivity chronocoulometric DNA sensor (CDS) based on gold nanoparticles (AuNPs)/poly(l-lysine) complex film modified glassy carbon electrode. Hexaammineruthenium(III) chloride ([Ru(NH₃)₆]³⁺) was used as the electroactive indicator. The assembled process was investigated by cyclic voltammetry (CV) and chronocoulometry (CC). CC is used to monitor the DNA hybridization event by measurement of electrostatic binding [Ru(NH₃)₆]³⁺. Under the optimal conditions, the signal of [Ru(NH₃)₆]³⁺ was linear with the logarithm of the concentration of the complementary oligonucleotides from 1.0 × 10⁻¹³ to 1.0 × 10⁻¹¹ M, and the detection limit is 3.5 × 10⁻¹⁴ M.     

Structural characterization reveals that viperin is a radical S-adenosyl-l-methionine (SAM) enzyme

Viperin is an interferon-inducible protein inhibiting many DNA and RNA viruses. It contains an N-terminal transmembrane helix, a highly conserved C-terminus and a middle region carrying a CX3CX2C motif, characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. So far no structural characterization has been reported and reconstitution of the [4Fe-4S] cluster in viperin all failed. Here, by dissecting the 361-residue human viperin into 12 fragments, followed by extensive CD and NMR characterization, Viperin (45-361) was identified to be soluble and structured in buffers. Most importantly, we have successfully reconstituted the [4Fe-4S] cluster in Viperin (45-361), thus providing the first experimental evidence confirming that viperin is indeed a radical SAM enzyme. Furthermore, the C-terminus Viperin (214-361) which is insoluble in buffers but again can be solubilized in salt-free water appears to be only partially folded. Our results thus imply that the radical SAM enzyme activity may play a key role in the broad antiviral actions of viperin.     

Selective recognition of d-tryptophan from d/l-tryptophan mixtures in the presence of Cu(II) by electropolymerized l-lysine film

Selective recognition of d-tryptophan (d-Trp) in the presence of Cu(II) was investigated at poly-l-lysine (p-l-Lys) film using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). p-l-Lys film was immobilized on a glassy carbon electrode (GCE) by cyclic voltammetry between 0.0 and 1.9 V in 20 mM phosphate buffer solution (pH 8.6). After the p-l-Lys/GCE electrode was incubated with d-Trp solution containing Cu(II) ions, obvious enhancement of electron transfer resistance and decrease of voltammetric current could be observed. If d-Trp was replaced by l-tryptophan (l-Trp), there was no apparent resistance and current changes. Moreover, no resistance and current changes could be found in the absence of Cu(II). It may be due to the formation of Cu complex with l-lysine and d-tryptophan. Finally, this method was successfully applied to monitoring enantiomeric composition of the d-Trp and l-Trp mixtures.     

Tuning one dimensional chiral channel interior in mixed ligand Cu(II) complexes of l-histidine derivative and substituted pyridine

Four pentacoordinated square-pyramidal Cu(II) complexes with the general formula [Cu(L)(X)], where L is a l-histidine derived tetradentate ligand and X is either 3-hydroxypyridine or 2-methylpyridine, has been synthesized. Structural analysis showed that the presence of water filled one dimensional chiral channel in the lattices. The interiors of the channels were varied using aromatic ring substitution on the ligand as well as on the monodentate ligand. The dimensions of the channels range from ∼7 to 9 Å.     

Preparation and characterisation of oxovanadium(IV) complexes derived from 2,6-diformyl-4-methylphenol and l-His and l-Ala. Spectroscopic study of the system VO + BDF-His

By reaction of 2,6-diformyl-4-methylphenol (BDF) with the amino acids l-His and l-Ala in the presence of VOCl₂, two new oxovanadium(IV) complexes with the ligand obtained by the 1:2 condensation of BDF with the amino acids, BDF-His and BDF-Ala, were synthesised. The compounds were characterised in the solid state by elemental analyses, IR, CD and magnetic susceptibility measurements, and in the mother liquor by EPR. In water-containing solutions, BDF-Ala and BDF-His partially hydrolyse but the degree of Schiff base formation increases upon addition of VOSO₄. The equilibria in the system V^IVO²⁺ + BDF-His were studied by spectroscopic methods (EPR, CD and UV-Vis) in the pH range 1.5-12. The coordination behaviour of the ligand changes as the pH increases, leading to the formation of four main species all involving O_phen as donor atom. Plausible binding modes are discussed based on the spectroscopic results.     

Kinetics and mechanism for reduction of halo- and haloam(m)ine platinum(IV) complexes by l-ascorbate

Reduction of the model platinum(IV) complexes cis-[PtCl₄(NH₃)₂] (1), trans-[PtCl₄(NH₃)₂] (2), trans-[PtCl₂(en)₂]²⁺ (3), trans-[PtBr₂(NH₃)₄]²⁺ (4), [PtCl₆]²⁻ (5), and [PtBr₆]²⁻ (6) with l-ascorbic acid (H₂Asc) in 1.0 M aqueous medium at 25 °C in the region 1.75≤pH≤7.20 has been investigated using stopped-flow spectrophotometry. The redox reactions follow the rate law: −d[Pt(IV]/dt=k[H₂Asc]_tot[Pt(IV)] where k is a pH-dependent second-order rate constant and [H₂Asc]_tot, the total concentration of ascorbic acid. The pH-dependence of k is attributed to parallel reduction of Pt(IV) by the protolytic species HAsc⁻ and Asc²⁻. Analysis of the kinetics data reveals that the ascorbate anion Asc²⁻ is up to seven orders of magnitude more reactive than HAsc⁻ while H₂Asc is unreactive. Electron transfer from HAsc⁻/Asc²⁻ to the Pt(IV) compounds is suggested to take place by a mechanism involving a reductive attack on any one of the mutually trans-halide ligands by Asc²⁻ and/or HAsc⁻ forming a halide-bridged activated complex. The rapid reduction of these complexes supports the assumption that ascorbate Asc²⁻ might be an important reductant at physiological conditions for anticancer active Pt(IV) pro-drugs capable of undergoing reductive trans elimination. The parameters ΔH^≠ and ΔS^≠ for reduction of Pt(IV) with Asc²⁻ have been determined from the study of the temperature dependence of k.     

Motor neuronal protection by l-arginine prolongs survival of mutant SOD1 (G93A) ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that l-arginine protects cultured motor neurons from excitotoxic injury. We also found that l-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, l-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that l-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.     

The efflux of l-carnitine from cells in culture (CCL 27)

The efflux of l-[³H]carnitine was studied in cells from an established cell line from human heart (Girardi human heart cells, CCL 27). The cells were loaded with 4 μmol/l l-[³H]carnitine for 1 or 24 h, and the efflux of radioactivity into the medium was measured. The amount of intracellular l-[³H]carnitine retained was expressed as a function of time. The results were fitted to an exponential equation, from which efflux rate constants were computed.Increasing the extracellular concentration of butyrobetaine, l-carnitine, d-carnitine, betaine, dl-norcarnitine or 3-dimethylamino-2-hydroxypropionic acid each increased the observed efflux. This is most likely due to accelerated exchange diffusion. The substrate specificity of this accelerated exchange diffusion is different from what previously has been found in competitive uptake studies of l-carnitine. l-Carnitine was preferentially released to l-acetylcarnitine, and blocking the sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) increased the efflux.     

Bis(pyrazolyl) palladium(II), platinum(II) and gold(III) complexes: Syntheses, molecular structures and substitution reactions with l-cysteine

A series of pyrazolyl palladium(II), platinum(II) and gold(III) complexes, [PdCl₂(3,5-R₂bpza)] {R = H (1), R = Me (2), bpza = bis-pyrazolyl acetic acid}, [PtCl₂(3,5-R₂bpza)] {R = H (3a), R = Me (4)}, [AuCl₂(3,5-R₂bpza)]Cl {R = H (5a), R = Me (6a)} and [PdCl₂(3,5-R₂bpzate)] {R = Me (7)} have been synthesised and structurally characterised. Single crystal X-ray crystallography showed that the pyrazolyl ligands exhibit N^N-coordination with the metals. Anticancer activities of six complexes 1-6a were investigated against CHO cells and were found to have low activities. Substitution reactions of selected complexes 1, 2, 3a and 5a with l-cysteine show that the low anticancer activities compounds and that the rate of substitution with sulfur-containing compounds is not the cause of the low anticancer activities.     

Effect of chronic l-DOPA treatment on 5-HT1A receptors in parkinsonian monkey brain

After chronic use of l-3,4-dihydroxyphenylalanine (l-DOPA), most Parkinson’s disease (PD) patients suffer from its side effects, especially motor complications called l-DOPA-induced dyskinesia (LID). 5-HT_1A agonists were tested to treat LID but many were reported to worsen parkinsonism. In this study, we evaluated changes in concentration of serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) and of 5-HT_1A receptors in control monkeys, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys, dyskinetic MPTP monkeys treated chronically with l-DOPA, low dyskinetic MPTP monkeys treated with l-DOPA and drugs of various pharmacological activities: Ro 61-8048 (an inhibitor of kynurenine hydroxylase) or docosahexaenoic acid (DHA) and dyskinetic MPTP monkeys treated with l-DOPA + naltrexone (an opioid receptor antagonist). Striatal serotonin concentrations were reduced in MPTP monkeys compared to controls. Higher striatal 5-HIAA/serotonin concentration ratios in l-DOPA-treated monkeys compared to untreated monkeys suggest an intense activity of serotonin axon terminals but this value was similar in dyskinetic and nondyskinetic animals treated with or without adjunct treatment with l-DOPA. As measured by autoradiography with [³H]8-hydroxy-2-(di-n-propyl) aminotetralin (8-OH-DPAT), a decrease of 5-HT_1A receptor specific binding was observed in the posterior/dorsal region of the anterior cingulate gyrus and posterior/ventral area of the superior frontal gyrus of MPTP monkeys compared to controls. An increase of 5-HT_1A receptor specific binding was observed in the hippocampus of MPTP monkeys treated with l-DOPA regardless to their adjunct treatment. Cortical 5-HT_1A receptor specific binding was increased in the l-DOPA-treated MPTP monkeys alone or with DHA or naltrexone and this increase was prevented in low dyskinetic MPTP monkeys treated with l-DOPA and Ro 61-8048. These results highlight the importance of 5-HT_1A receptor alterations in treatment of PD with l-DOPA.     

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule,CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration

Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (K_m 0.24 μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.