Structure-activity relationship of celecoxib and rofecoxib for the membrane permeabilizing activity.

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory effect by inhibiting cyclooxygenase activity. We previously suggested that in addition to cyclooxygenase-inhibition at the gastric mucosa, NSAID-induced gastric mucosal cell death is required for the formation of NSAID-induced gastric lesions in vivo. We showed that celecoxib exhibited the most potent membrane permeabilizing activity among the NSAIDs tested. In contrast, we have found that the NSAID rofecoxib has very weak membrane permeabilizing activity. To understand the membrane permeabilizing activity of coxibs in terms of their structure-activity relationship, we separated the structures of celecoxib and rofecoxib into three parts, synthesized hybrid compounds by substitution of each of the parts, and examined the membrane permeabilizing activities of these hybrids. The results suggest that the sulfonamidophenyl subgroup of celecoxib or the methanesulfonylphenyl subgroup of rofecoxib is important for their potent or weak membrane permeabilizing activity, respectively. These findings provide important information for design and synthesis of new coxibs with lower membrane permeabilizing activity.

Evasion of the accelerated blood clearance phenomenon by coating of nanoparticles with various hydrophilic polymers.

The accelerated blood clearance (ABC) phenomenon is induced upon repeated injections of poly(ethylene glycol) (PEG)-coated colloidal carriers. It is essential to suppress this phenomenon in a clinical setting because the pharmacokinetics must be reproducible. In this study, we evaluated the induction of the ABC phenomenon using nanoparticles coated with various hydrophilic polymers instead of PEG. Nanoparticles encapsulating prostaglandin E1 were prepared by the solvent diffusion method from a blend of poly(lactic acid) (PLA) and block copolymers consisting of various hydrophilic polymers and PLA. Coating of nanoparticles with poly(N-vinyl-2-pyrrolidone) (PVP), poly(4-acryloylmorpholine), or poly(N,N-dimethylacrylamide) led to extended residence of the nanoparticles in blood circulation in rats, although they had a shorter half-life than the PEG-coated nanoparticles. The ABC phenomenon was not induced upon repeated injection of PVP-coated nanoparticles at various time intervals, dosages, or frequencies, whereas it was elicited by PEG-coated nanoparticles. In addition, anti-PVP IgM antibody, which is estimated to be one of the crucial factors for induction of the ABC phenomenon, was not produced after injection of PVP-coated nanoparticles. These results suggest that the use of PVP, instead of PEG, as a coating material for colloidal carriers can evade the ABC phenomenon.

Synthesis of prostaglandin E(1) phosphate derivatives and their encapsulation in biodegradable nanoparticles.

PURPOSE: Prostaglandin E(1) (PGE(1)) is an effective treatment for peripheral vascular diseases. The encapsulation of PGE(1) in nanoparticles for its sustained-release would improve its therapeutic effect and quality of life (QOL) of patients. METHODS: In order to encapsulate PGE(1) in nanoparticles prepared with a poly(lactide) homopolymer (PLA) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA), we synthesized a series of PGE(1) phosphate derivatives and tested their efficacy. RESULTS: Among them, PGE(1) 2-(phosphonooxy)ethyl ester sodium salt (C2) showed the most efficient hydrolysis to yield PGE(1) in human serum. An in vitro platelet aggregation assay showed that C2 inhibited aggregation only after pre-incubation in serum, suggesting that C2 is a prodrug of PGE(1). In vivo, intravenous administration of C2 caused increase in cutaneous blood flow. In the presence of zinc ions, all of the synthesized PGE(1) phosphate derivatives could be encapsulated in PLA-nanoparticles. Use of L-PLA instead of D,L-PLA, and high molecular weight PLA resulted in a slower release of C2 from the nanoparticles. CONCLUSIONS: We consider that C2-encapsulated nanoparticles prepared with L-PLA and PEG-D,L-PLA have good sustained-release profile of PGE(1), which is useful clinically.

Accelerated blood clearance phenomenon upon repeated injection of PEG-modified PLA-nanoparticles.

PURPOSE: We recently developed prostaglandin E(1) (PGE(1))-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw = 17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats. METHODS: The plasma levels of PGE(1) and anti-PEG IgM antibody were determined by EIA and ELISA, respectively. RESULTS: Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw = 28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE(1) than NP-L20. CONCLUSIONS: This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE(1).

Simultaneous measurements of K+ and calcein release from liposomes and the determination of pore size formed in a membrane.

The changes induced by biologically active substances in the permeability to K+ and calcein of liposomes composed of egg phosphatidylcholine and cholesterol were measured simultaneously in order to rapidly screen the sizes of pores formed in a membrane, using different sized markers. The substances examined in the present study were classified into three types based on differences in the rates at which K+ and calcein were released. The first type released only K+, and included gramicidin A. The second type predominantly released K+, preceding the release of calcein, and included amphotericin B and nystatin. The third type, including antimicrobial peptides, such as gramicidin S, alamethicin, and melittin, and several membrane-active drugs, like celecoxib (non-steroidal anti-inflammatory drug), 1-dodecylazacycloheptan-2-one (named azone; skin permeation enhancer), and chlorpromazine (tranquilizer), caused the release of K+ and calcein simultaneously. Thus, the sizes of pores formed in a liposomal membrane increased in the following order: types one, two, and three. We determined the size more precisely by conducting an osmotic protection experiment, measuring the release of calcein in the presence of osmotic protectants of different sizes. The radii of pores formed by the second type, amphotericin B and nystatin, were 0.36 - 0.46 nm, while the radii of pores formed by the third type were much larger, 0.63 - 0.67 nm or more. The permeability changes induced by substances of the third type are discussed in connection with a transient pore formed in a lipid packing mismatch taking place during the phase transition of dipalmitoylphosphatidylcholine liposomes.

Encapsulation of beraprost sodium in nanoparticles: analysis of sustained release properties, targeting abilities and pharmacological activities in animal models of pulmonary arterial hypertension.

Prostaglandin I2 (PGI2) and its analogues (such as beraprost sodium, BPS) are beneficial for the treatment of pulmonary arterial hypertension (PAH). The encapsulation of BPS in nanoparticles to provide sustained release and targeting abilities would improve both the therapeutic effect of BPS on PAH and the quality of life of patients treated with this drug. BPS was encapsulated into nanoparticles prepared from a poly(lactic acid) homopolymer and monomethoxy poly(ethyleneglycol)-poly(lactide) block copolymer. The accumulation of nanoparticles in damaged pulmonary arteries was examined using fluorescence-emitting rhodamine S-encapsulated nanoparticles. The monocrotaline-induced PAH rat model and the hypoxia-induced mouse model were used to examine the pharmacological activity of BPS-encapsulated nanoparticles. A nanoparticle, named BPS-NP, was selected among various types of BPS-encapsulated nanoparticles tested; this was based on the sustained release profile in vitro and blood clearance profile in vivo. Fluorescence-emitting rhodamine S-encapsulated nanoparticles were prepared in a similar manner to that of BPS-NP, and showed accumulation and prolonged residence in monocrotaline-damaged pulmonary peripheral arteries. Intravenous administration of BPS-NP (once per week, 20µg/kg) protected against monocrotaline-induced pulmonary arterial remodeling and right ventricular hypertrophy. The extent of this protection was similar to that observed with oral administration (once per day, 100µg/kg) of BPS alone. The once per week intravenous administration of BPS-NP (20µg/kg) also exhibited an ameliorative effect on hypoxia-induced pulmonary arterial remodeling and right ventricular hypertrophy. The beneficial effects of BPS-NP on PAH animal models seem to be mediated by its sustained release and tissue targeting profiles. BPS-NP may be useful for the treatment of PAH patients due to reduced dosages and frequency of BPS administration.

Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli.

A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.

Development of biodegradable nanoparticles for liver-specific ribavirin delivery.

Ribavirin is an antiviral drug used for the treatment of chronic hepatitis C. However, ribavirin induces severe side effects such as hemolytic anemia. In this study, we prepared biodegradable nanoparticles as ribavirin carriers to modulate the pharmacokinetics of the drug. The nanoparticles encapsulating ribavirin monophosphate (RMP) were prepared from the blend of poly(d,l-lactic acid) homopolymer and arabinogalactan (AG)-poly(l-lysine) conjugate by using the solvent diffusion method in the presence of iron (III). RMP was efficiently and stably embedded in the nanoparticles and gradually released for 37 days in phosphate-buffered saline at 37°C. The coating of AG on the nanoparticles surfaces was verified by measuring the zeta potentials and performing an aggregation test of the nanoparticles using galactose-binding lectin. Moreover, the nanoparticles were efficiently internalized in cultured HepG2 cells. Ribavirin was drastically accumulated to the liver of mice after intravenous administration of the RMP-loaded nanoparticles, after which the ribavirin content gradually decreased for at least 7 days. Our results indicated successful development of nanoparticles with dual functions, targeting to the liver and sustained release of ribavirin, and suggested that the present strategy could help to advance the clinical application of ribavirin as a therapeutic agent for chronic hepatitis C.

Techniques for efficient entrapment of pharmaceuticals in biodegradable solid micro/nanoparticles.

IMPORTANCE OF THE FIELD: Biodegradable solid particles are potential carriers for both hydrophobic and hydrophilic drugs and have been marketed for prolongation of pharmaceutical activity. In developing such particles, it is important to achieve stable encapsulation of the drugs in the particles and a controlled rate of drug release. AREAS COVERED IN THIS REVIEW: In this paper, the principles and techniques for preparing such particles are reviewed. WHAT THE READER WILL GAIN: Overall, it remains difficult to identify a general approach for achieving effective entrapment and controlled release, because these qualities are determined by multiple complex factors. The encapsulation efficiency of drugs in particles can be improved through various techniques, including hydrophobic interaction, covalent bonding, ionic interaction and physical isolation. In addition, the release behaviors of drugs are strongly influenced by environmental conditions as well as the physicochemical properties of the polymers and drugs. TAKE HOME MESSAGE: Solid particles with targeting ability in addition to prolongation of biological activity may have potential for development of a new type of pharmaceutical in the clinical field.

Preparation and characterization of a nanoparticulate formulation composed of PEG-PLA and PLA as anti-inflammatory agents.

We have prepared polymeric nanoparticles using a blend of poly(lactic acid) and monomethoxy-polyethyleneglycol(PEG)-polylactide block copolymer along with betamethasone disodium phosphate (BP). Nanoparticles have been screened for anti-inflammatory activity using experimental rat models of inflammation. In the present study, we examined the degradation of nanoparticles in vitro during incubation. We found that the nanoparticles lost the PEG chains present on their surfaces within a few days, and subsequently gradually released BP. Furthermore, we found that these nanoparticles preferentially accumulated in the inflammatory lesion in adjuvant arthritis rat models, and that the amount of BP gradually depleted from the lesion over 14 days. These results suggested the mechanism underlying the anti-inflammatory effect of the nanoparticles in vivo: the initial accumulation of BP in the lesion due to the enhanced permeability and retention effect, the subsequent internalization in inflammatory macrophages due to the loss of PEG, and the release of BP in cells during the hydrolysis of polymers. The nanoparticles were successfully prepared on a large-scale and stably stored in the form of a freeze-dried formulation for at least 69 weeks below 25 degrees C. These results suggest that the nanoparticles can be used as an anti-inflammatory pharmaceutical formulation in a clinical setting.

Prolonging the in vivo residence time of prostaglandin E(1) with biodegradable nanoparticles.

PURPOSE: Prostaglandins have potent and diverse biologic activities, but their clinical application is severely restricted, mainly due to rapid inactivation in vivo. In order to modulate the pharmacokinetics of prostaglandin E(1) (PGE(1)), we prepared biodegradable nanoparticles as a drug carrier. METHODS: Nanoparticles encapsulating PGE(1) were prepared from a blend of poly(lactic acid) homopolymer and poly(ethylene glycol)-poly(lactide) block copolymer by the solvent diffusion method in the presence of iron. RESULTS: PGE(1) was efficiently and stably embedded in the nanoparticles through interaction with iron, despite being relatively hydrophilic and having unstable chemical properties. Depending on the isomers and molecular weight of poly(lactic acid) selected, PGE(1) was gradually released from the nanoparticles at various rates into diluted serum in vitro. Both stable retention of PGE(1) in the nanoparticles and coating of the nanoparticles with poly(ethylene glycol) led to an extremely extended blood residence time of PGE(1), as well as preferential accumulation in vascular lesions. CONCLUSIONS: These results suggest that the present strategy is useful to advance the clinical application of PGE(1) as a therapeutic agent for vascular disorders.

Involvement of intracellular Ca2+ levels in nonsteroidal anti-inflammatory drug-induced apoptosis.

We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells.

Membrane permeabilization by non-steroidal anti-inflammatory drugs.

The cytotoxicity of non-steroidal anti-inflammatory drugs (NSAIDs) is involved in the formation of NSAID-induced gastric lesions. The mechanism(s) behind these cytotoxic effects, however, is not well understood. We found here that several NSAIDs tested caused hemolysis when employed at concentrations similar to those that result in cytotoxicity. Moreover, these same NSAIDs were found to directly permeabilize the membranes of calcein-loaded liposomes. Given the similarity in NSAID concentrations for cytotoxic and membrane permeabilization effects, the cytotoxic action of these NSAIDs may be mediated through the permeabilization of biological membranes. Increase in the intracellular Ca(2+) level can lead to cell death. We here found that all of NSAIDs tested increased the intracellular Ca(2+) level at concentrations similar to those that result in cytotoxicity. Based on these results, we consider a possibility that membrane permeabilization by NSAIDs induces cell death through increase in the intracellular Ca(2+) level.