Solubility and Physical Stability Enhancement of Loratadine by Preparation of Co-Amorphous Solid Dispersion with Chlorpheniramine and Polyvinylpyrrolidone.

Loratadine (LRD), a non-sedating and slow-acting antihistamine, is often given in combination with short-onset chlorpheniramine maleate (CPM) to increase efficacy. However, LRD has poor water solubility resulting in low bioavailability. The aim of this study was to improve LRD solubility by preparing co-amorphous LRD-CPM. However, the obtained co-amorphous LRD-CPM recrystallized rapidly, and the solubility of LRD returned to a poor state again. Therefore, co-amorphous LRD-CPM solid dispersions using polyvinylpyrrolidone (PVP) as a carrier were prepared. The obtained solid dispersions were characterized using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FT-IR). The solubility, dissolution, and mechanism of drug release from the LRD-CPM/PVP co-amorphous solid dispersions were studied and compared with those of intact LRD, LRD/PVP solid dispersions, and co-amorphous LRD-CPM mixtures. The results from XRPD and DSC confirmed the amorphous form of LRD in the co-amorphous solid dispersions. The FTIR results indicated that there was no intermolecular interaction between LRD, CPM, and PVP. In conclusion, the obtained LRD-CPM/PVP co-amorphous solid dispersions can successfully increase the water solubility and dissolution of LRD and extend the amorphous state of LRD without recrystallization.

Thermosensitive Polymer Blend Composed of Poloxamer 407, Poloxamer 188 and Polycarbophil for the Use as Mucoadhesive In Situ Gel.

Herein, thermosensitive blends of poloxamer 407 (P407)/poloxamer 188 (P188)/polycarbophil (PCB) were developed in terms of maximized content of PCB (a mucoadhesive polymer) and desired temperature-dependent rheological properties of the blends as in situ gelling matrices. Maximizing PCB content while achieving the preferable rheological characteristics was accomplished through the Box-Behnken design. The quantitative effect of the polymer composition in the blends on the thermosensitive characteristics was evaluated using the fitted design model and the corresponding surface plots. The optimized P407/P188/PCB blend (OPT) was the mixture of 20.000, 7.349 and 0.595% (w/w) of P407, P188, and PCB, respectively. The thermosensitive micellization of OPT was investigated using differential scanning calorimetry which revealed an overlapping double endothermic peak caused by the temperature-induced micellization of pure micelles in co-existence with the micelles with attached PCB. Mixing PCB with the P407/P188 matrix promoted a more intense mucoadhesion of the blend. After incorporating metronidazole, a model hydrophilic drug, into OPT, the temperature-dependent characteristics of the hydrogel did not change. Metronidazole release from OPT was sustained by an anomalous mechanism. This optimal ternary hydrogel benefiting from thermosensitive gelling and mucoadhesive matrix might be used as a viable platform for mucoadhesive in situ gelling drug delivery.

Syntheses and crystal structures of hydrated and anhydrous 1:2 cocrystals of oxyresveratrol and zwitterionic proline.

The hydrated and anhydrous 1:2 cocrystals of oxyresveratrol (4-[(E)-2-(3,5-di-hydroxy-phen-yl)ethen-yl]benzene-1,3-diol; OXY; C14H12O4) and proline [(S)-pyrrolidine-2-carb-oxy-lic acid; PRO; C5H9NO2], namely, 4-[(E)-2-(3,5-di-hydroxy-phen-yl)ethen-yl]benzene-1,3-diol bis-[(S)-pyrrolidin-1-ium-2-carboxyl-ate] monohydrate, C14H12O4·2C5H9NO2·H2O, and the anhydrous form, C14H12O4·2C5H9NO2, were obtained by crystallization at different temperatures. Both of them crystallize with ortho-rhom-bic (P212121) symmetry. The structures display N-H⋯O and O-H⋯O hydrogen-bonding inter-actions between PRO and PRO, OXY and OXY, and OXY and PRO. In the hydrated cocrystal, these types of contacts are also observed between the OXY, PRO and water mol-ecules. A combination of these inter-actions leads to a three-dimensional supra-molecular assembly in each case. Hirshfeld surfaces were used to gain further insight into the inter-molecular inter-actions in the packing, including the relative percentage contributions of the significant inter-molecular H⋯H and H⋯O/O⋯H contacts.

Exploring potential coformers for oxyresveratrol using principal component analysis.

Oxyresveratrol (OXY) exhibits poor bioavailability due to its low solubility. Cocrystals are effective in improving solubility of OXY. In this study, principal component analysis (PCA) was employed for coformer selection using 5 variables (solubility parameter, logP, hydrogen bonding acceptor, hydrogen bonding donor and molecular volume). According to PCA analyses, nicotinamide (NCT) and proline (PRO) were grouped as the potential coformers. OXY-PRO and OXY-NCT cocrystals were obtained using a liquid assisted grinding method. The thermal and diffraction characteristics of both cocrystals were different from their starting materials. Based on FTIR and Raman spectroscopy, both interactions between the amide group of NCT and the phenolic hydroxyl groups of OXY, as well as π-π interactions between the aromatic rings of OXY and the pyridine ring of NCT were involved in the cocrystal formation. For OXY-PRO cocrystal, the carboxylate group of PRO interacted with the hydroxyl groups of OXY, OXY-NCT and OXY-PRO cocrystals enhanced the solubility and dissolution of OXY. Accordingly, PCA proved to be an effective technique in screening potential coformers for OXY and probably for other compounds. The improved solubility and dissolution of OXY in the form of cocrystal may provide a benefit for the use of OXY for pharmaceutical applications.

Thermosensitive Poloxamer 407/Poly(Acrylic Acid) Hydrogels with Potential Application as Injectable Drug Delivery System.

Thermosensitive hydrogels are of great interest for in situ gelling drug delivery. The thermosensitive vehicle with a gelation temperature in a range of 30-36°C would be convenient to be injected as liquid and transform into gel after injection. To prepare novel hydrogels gelling near body temperature, the gelation temperature of poloxamer 407 (PX) were tailored by mixing PX with poly(acrylic acid) (PAA). The gelation behaviors of PX/PAA systems as well as the interaction mechanism were investigated by tube inversion, viscoelastic, shear viscosity, DSC, SEM, and FTIR studies. The gelation temperature of the plain PX solutions at high concentration of 18, 20, and 22% (w/w) gelled at temperature below 28°C, which is out of the suitable temperature range. Mixing PX with PAA to obtain 18 and 20% (w/w) PX with 1% (w/w) PAA increased the gelation temperature to the desired temperature range of 30-36°C. The intermolecular entanglements and hydrogen bonds between PX and PAA may be responsible for the modulation of the gelation features of PX. The mixtures behaved low viscosity liquid at room temperature with shear thinning behavior enabling their injectability and rapidly gelled at body temperature. The gel strength increased, while the pore size decreased with increasing PX concentration. Metronidazole, an antibiotic used for periodontitis, was incorporated into the matrices, and the drug did not hinder their gelling ability. The gels showed the sustained drug release characteristic. The thermosensitive PX/PAA hydrogel could be a promising injectable in situ gelling system for periodontal drug delivery.

Microphase Separation and Gelation of Methylcellulose in the Presence of Gallic Acid and NaCl as an In Situ Gel-Forming Drug Delivery System.

Novel hydrogels of methylcellulose (MC) with gallic acid (GA) and NaCl were developed for an in situ gel-forming delivery system. Plain MC and GA/NaCl/MC were characterized using micro-differential scanning calorimetry (micro-DSC), rheological and turbidity methods. The gelation temperatures of MC were reduced to body temperature with adding GA/NaCl. GA and NaCl caused slightly different effects on the gelation/degelation temperatures during heating/cooling, respectively, based on the different sensitivities of these three techniques. The gelation mechanism was investigated by UV spectrophotometry, and the hydrophobic interaction between the aromatic ring of GA and MC was verified. The NaCl/MC hydrogel had smaller micropores than GA/MC and MC, indicating a greater cross-linked density. Doxycycline (DX) was loaded into the systems and demonstrated a synergistic effect of DX/GA. Both GA and DX exhibited a sustained release. The hydrogel of GA/4NaCl/MC could be potentially used for the in situ delivery of DX for deep wound healing.

Fabrication of pluronic and methylcellulose for etidronate delivery and their application for osteogenesis.

Novel hydrogels were prepared by blending 4% (w/w) methylcellulose (MC) with various concentrations of 12, 14, 16, 18 and 20% (w/w) pluronic F127 (PF) to form injectable implant drug delivery systems. The blends formed gels using lower concentrations of PF compared to when using PF alone. Etidronate sodium (EDS) at a concentration of 4×10(-3)M was loaded into these blends for producing an osteogenesis effect. The pure gels or EDS loaded gels exhibited cytocompatibility to both the osteoblast (MC3T3-E1) and myoblast (C2C12) cell lines whereas the gels of 16PF, 18PF and 20PF were very cytotoxic to the cells. The EDS loaded gels demonstrated significantly greater alkaline phosphatase (ALP) activities compared to the pure gels. The longer exposure time periods of the samples to the cells, the greater was the ALP activity. These EDS loaded gels significantly increased proliferation of both cell lines thus indicating a bone regeneration effect. The PF/MC blends prolonged the in vitro release of EDS for more than 28 days. Based on the in vitro degradation test, the MC extensively improved the gel strength of the PF and delayed the degradation of the gels thus making them more functional for a sustained drug delivery for osteogenesis.

Characterization of supramolecular gels based on ß-cyclodextrin and polyethyleneglycol and their potential use for topical drug delivery.

Novel gels were prepared by blending ß-cyclodextrin and polyethyleneglycol (PEG) in the presence of K2CO3. The objective of this study was thus to characterize the gels using rheology, modulated temperature differential scanning calorimetry (MTDSC), turbidity measurements, and hot stage microscopy, and then investigate the potential use of the gel for topical drug delivery. Two types of supramolecular gels, GelL and GelH were prepared at a low temperature (below 50 °C) and at a high temperature (above 70 °C), respectively. Both gels were thermo-reversible. Upon heating, GelL could turn to GelH. Nevertheless, upon cooling, GelH that was more stable than GelL precipitated and GelL could not be reformed. GelL may form through simple complexation of polyethyleneglycol (PEG) with ß-cyclodextrin in the presence of K2CO3. However, GelH may form a specific complex or a pseudopolyrotaxane gel. For pharmaceutical application, GelL was investigated instead of GelH because the forming temperature of this gel was close to the human body temperature. The interactions among diclofenac sodium (DS), a model drug, and the components of the gel were examined using FTIR. These interactions may include ionic attraction and hydrogen bonds between the carboxylate groups of DS and the hydroxyl groups of PEG or ß-cyclodextrin and probably also the inclusion of the aromatic ring of DS into the cavity of ß-cyclodextrin. Furthermore, the release and permeation of diclofenac from GelL were significantly greater than those from a commercial gel. Therefore, GelL may be useful for the topical delivery of drugs.

Characterization of freeze-dried gallic acid/xyloglucan.

BACKGROUND: Tamarind seed xyloglucan (TSX) is generally used for drug delivery systems. Gallic acid (GA) possesses various pharmacological activities. It has a good solubility and bioavailability but short half-life. PURPOSE: To prepare a sustained-release of GA to overcome its relatively short half-life. GA was blended with TSX and freeze-dried. The physicochemical properties of freeze-dried GA and freeze-dried GA/TSX were characterized, and the release profiles of GA from these freeze-dried samples were investigated. METHOD: All freeze-dried samples were characterized by PXRD, spectroscopic and thermal analyses. The dissolution studies were performed according to the United States Pharmacopeia (USP) XXX. RESULTS: According to FTIR, FT-Raman and (13)C CP/MAS NMR, the spectra of freeze-dried GA were similar to that of the anhydrous form. Nevertheless, DRIFTS and DSC were able to differentiate these two forms. The crystallinity of GA in the freeze-dried GA/TSX was the same as that of the freeze-dried GA. DSC indicates that there were interactions between GA and TSX. It was of interest that a freeze-dried sample with low amount of GA, 0.2% GA/1% TSX was mostly in an amorphous form. Moreover, all freeze-dried GA/TSX preparations demonstrated a sustained-release of GA compared to GA alone. The freeze-dried 1% GA/1% TSX provided the best sustained-release of GA of up to 240 min. CONCLUSIONS: TSX could change a crystal form of a small molecule to a mostly amorphous form. It was of importance that the freeze-dried GA/TSX could effectively retard the release of GA. These samples may be able to overcome the limitation for the therapeutic use of GA due to its short biological half-life.

Conformations and spectroscopic properties of laccaic acid A in the gas phase and in implicit water.

Conformations and spectroscopic properties of laccaic acid A (lacA) were studied by means of the experimental and theoretical approaches. The minimum energy conformers of lacA in the gas phase and in implicit water obtained from the B3LYP/6-311G(d,p) calculations displayed the same orientation of the COOH and OH groups on the anthraquinone-based component. The intramolecular hydrogen bonds (H-bonds) formed between the COOH, C=O and OH groups are very strong. In contrast, the orientations of the Ph(OH)CH(2)CH(2)NHCOCH(3) substituent moiety on the anthraquinone-based component in the gas phase and in implicit water are completely different. The substituent prefers to bind with the anthraquinone-based component in the gas phase while it moves away from the anthraquinone-based component in implicit water. The calculated IR spectra of the two lowest-lying energy conformers of lacA in the gas phase fit to the experimental FTIR spectrum. The full assignments of the vibrational modes with the correlated vibrational wavenumbers of those conformers were proposed here, for the first time. The intramolecular H-bond formations in lacA can cause the shift of the vibrational wavenumber for the COOH, C=O, OH and NH groups as compared to the normal vibrations of these groups. The NMR spectra showed that the stabilities of the two lowest-lying energy conformers of lacA in the gas phase are comparable and this is consistent with their computational energies. The UV-Vis spectra of the lowest-lying energy conformers of lacA in implicit water were compared with the experimental UV-Vis spectrum. The calculations suggested that the electronic transition in the visible region involves with the singlet π→π(*) excitation which the electron density transfers to a COOH group on the anthraquinone ring.

SAXS and ATR-FTIR studies on EBT-TSX mixtures in their sol-gel phases.

Our previous study demonstrated that mixtures of tamarind seed xyloglucan (TSX) with appropriate concentrations of eriochrome black T (EBT) produced a gel that could be of benefit for medical use. Here, the sol-gel systems of various fresh and aged mixtures were further investigated using rheological measurements. The nanostructural changes of EBT-TSX sol-gel phases were analyzed using SAXS. The interactions between EBT and TSX in the sol and gel states were examined using ATR-FTIR. SAXS data analysis demonstrated that the mixture containing lower concentration of EBT formed rod-like structures and that with higher concentrations of EBT produced flat particles. The sizes of the TSX structures from the aged mixtures in the gel stage were larger than those from the same mixtures in the sol state. ATR-FTIR spectral changes revealed that the azo and sulfonic acid groups of EBT interacted with the TSX, and the characteristic spectrum of the sulfonic acid group of EBT could discriminate between the sol and gel state of the EBT-TSX systems. The interactions between EBT and TSX may cause conformational changes to TSX and facilitate the sol-gel transition or formation of a gel.

Glabridin.

In the title compound, C20H20O4 {systematic name: 4-[(3R)-8,8-dimethyl-3,4-dihydro-2H-pyrano[2,3-f]chromen-3-yl]benz-ene-1,3-diol}, the hydro-pyran ring linked to the pendant benzene ring adopts an envelope conformation, with the methyne C atom forming the flap. In the crystal, the -OH group at the 3-position of the benzene ring forms an O-H⋯O hydrogen bond to a chromene O-atom acceptor, whereas the -OH group at the 1-position forms an O-H⋯π inter-action with a neighboring benzene ring. The O-H⋯O hydrogen bonds form [001] chains and the O-H⋯π bonds cross-link the chains into (101) sheets. The absolute structure was assumed to be the same as that deduced from previous studies for the natural product.

Experimental FTIR and theoretical studies of gallic acid-acetonitrile clusters.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) has many possible conformers depending on the orientations of its three OH and COOH groups. The biological activity of polyphenolic compounds has been demonstrated to depend on their conformational characteristics. Therefore, experimental FTIR and theoretical studies of the GA-solvent clusters were performed to investigate the possible most favored conformation of GA. Acetonitrile (ACN) was selected as the solvent since its spectrum did not interfere with the OH stretching bands of GA. Also of importance was that these OH groups, in addition to the carboxyl group, of the GA are the most likely groups to interact with receptors. The solution of GA in the ACN solution was measured and the complex OH bands were deconvoluted to four component bands. These component bands corresponded to the three OH bands on the benzene ring and a broad band which is a combination band of mainly the OH of the COOH group and the inter- and intramolecular H-bonds from the OH groups on the ring. The conformations, relative stabilities and vibrational analysis of the GA monomers and the GA-ACN clusters were investigated using the B3LYP/6-311++G(2d,2p) method. Conformational analysis of the GA monomer yielded four most possible conformers, GA-I, GA-II, GA-III and GA-IV. These conformers were subsequently used for the study of the GA:ACN clusters at the 1:1, 1:2 and 1:4 mole ratios. The IR spectra of the most stable structures of these clusters were simulated and the vibrational wavenumbers of the OH and C=O groups were compared with those from the experiment. The FTIR component bands were comparable to the computed OH bands of the GA-I-(ACN)(2), GA-IV-(ACN)(2) and GA-I-(ACN)(4) clusters. Furthermore, the C=O stretching bands and the bands in the regions of 1800-1000 cm(-1) obtained by computing and the experiment were similar for these clusters. Thus, GA-I and GA-IV are the most preferable conformations of GA in ACN and perhaps in the polar environment around the receptor sites of GA.

3,4,5-Trihy-droxy-benzoic acid.

In the title compound, C(7)H(6)O(5), the three hy-droxy groups on the ring are oriented in the same direction. There are two intra-molecular O-H⋯O hydrogen bonds in the ring. In the crystal, there are several inter-molecular O-H⋯O hydrogen bonds and a short contact of 2.7150 (18) Šbetween the O atoms of the para-OH groups of adjacent mol-ecules.

Characterization of muco- and bioadhesive properties of chitosan, PVP, and chitosan/PVP blends and release of amoxicillin from alginate beads coated with chitosan/PVP.

PURPOSE: To investigate the muco/bioadhesive properties of chitosan, polyvinylpyrrolidone (PVP), and chitosan/PVP blends and the release of amoxicillin (AMX) contained in AMX-alginate beads coated with these materials. METHOD: Chitosan, PVP, and chitosan/PVP blends at various volume ratios were coated onto calcium alginate beads containing AMX. The muco/bioadhesive properties of all materials and the AMX-alginate beads coated with these materials were characterized. RESULTS: Measurements of their viscosity, texture, and adhesion to HT29 cells demonstrated that chitosan/PVP at a volume ratio of 5/5 had the best muco/bioadhesive properties when compared with chitosan, PVP, and blends of other ratios. Wash-off tests indicated that the mucoadhesive property of the coated AMX-alginate beads was significantly higher than that of the uncoated beads. Diffuse reflectance infrared Fourier transform spectroscopy showed that there were interactions between chitosan-PVP, chitosan-mucin, PVP-mucin, and chitosan/PVP blend-mucin. Scanning electron microscopy revealed that the surfaces of the coated beads were smoother than those of the uncoated beads. All coated AMX-alginate beads were able to provide a controlled release of AMX with Super Case II transport properties, at a pH of 4. This was probably a result of the rapid and extensive swelling of the alginate beads. The more rapid release of AMX at pH 1 was probably because of the rapid dissolution of the drug at this pH. CONCLUSIONS: From the controlled drug release and muco/bioadhesive properties of these coated AMX-alginate beads, we suggest that the alginate-coated beads might be a promising drug delivery system to assist with the eradication of Helicobacter pylori infections.

Characterization of cimetidine-piroxicam coprecipitate interaction using experimental studies and molecular dynamic simulations.

The crystalline states of cimetidine and piroxicam, when coprecipitated from solvents containing 1:1 mole ratio, were transformed to amorphous states as observed using powder X-ray diffraction (PXRD). Amorphous forms of drugs generally exhibit higher water solubility than crystalline forms. It is therefore interesting to investigate the interactions that cause the transformation of both the crystalline drugs. Intermolecular interactions between the drugs were determined using Fourier-transform infrared spectroscopy (FTIR) and solid-state (13)C CP/MAS NMR. Molecular dynamic (MD) simulation was performed for the first time for this type of study to indicate the specific groups involved in the interactions based on radial distribution function (RDF) analyses. RDF is a useful tool to describe the average density of atoms at a distance from a specified atom. FTIR spectra revealed a shift of the C identical withN stretching band of cimetidine. The (13)C CP/MAS NMR spectra indicated downfield shifts of C(11), C(15) and C(7) of piroxicam. RDF analyses indicated that intermolecular interactions occurred between the amide oxygen atom as well as the pyridyl nitrogen of piroxicam and H-N(3) of cimetidine. The hydrogen atom (O-H) at C(7) interacts with the N(1) of cimetidine. Since the MD simulation results are consistent with, and complementary to the experimental analyses, such simulations could provide a novel strategy for investigating specific interacting groups of drugs in coprecipitates, or in amorphous mixtures.

Effect of Eriochrome Black T on the gelatinization of xyloglucan investigated using rheological measurement and release behavior of Eriochrome Black T from xyloglucan gel matrices.

A novel gel system was obtained by mixing aqueous solutions of tamarind seed xyloglucan (TSX) and Eriochrome Black T (EBT), an antiangiogenic compound. The shear-viscosity flow curves revealed that all the studies mixtures displayed a shear thinning behavior. Viscosity increased with increasing EBT concentrations. According to frequency sweep tests, mixtures at EBT concentration of 1.30% and 2.50% (w/v) in 1% (w/v) TSX formed a weak gel. The time sweep tests revealed that these mixtures remained as sol at room temperature (25 degrees C) for a long period of time but turned into gel in a short time at body temperature (37 degrees C). The in vitro EBT release profiles demonstrated sustained release of EBT. Loading concentration of EBT affected the gel strength and consequently the release mechanism of EBT. According to release kinetic analyses, the release profiles of 1.30% and 2.50% (w/v) EBT systems occur through an anomalous mechanism and Fickian diffusion, respectively. In conclusion, these EBT-TSX systems appear to be suitable as injectable implants for sustained delivery of EBT at a site of application, and as such they may be beneficial for the future treatment of solid malignant tumors.

Crystal structure transformations and dissolution studies of cimetidine-piroxicam coprecipitates and physical mixtures.

We have recently demonstrated that coprecipitation of cimetidine (C) and piroxicam (P) at a mole ratio of 1:1 results in the transformation of the crystalline forms of both drugs to an amorphous state. In this study, coprecipitates and physical mixtures of cimetidine and piroxicam were further investigated at C/P mole ratios of 1:10, 1:5, 1:4, 1:2, 10:1, 20:1, 30:1, 40:1, and 52.5:1, the latter being the composition of a clinically used dosage. The physicochemical properties of these samples were examined using X-ray diffraction and Fourier transform infrared spectroscopy. Additionally, dissolution of piroxicam in the samples at C/P mole ratios of 10:1, 20:1, 30:1, 40:1, and 52.5:1 was investigated at pH 1.2 and pH 4. In coprecipitates with C/P mole ratios of 10:1, 20:1, 30:1, and 40:1, crystalline forms of both drugs were transformed to amorphous states. A mixture of an amorphous state and cimetidine crystalline form A was observed for the coprecipitate with a C/P mole ratio of 52.5:1. For the coprecipitates with C/P mole ratios of 1:2, 1:4, 1:5, and 1:10, cimetidine form A was transformed to form C, whereas piroxicam form II was modified to form I. It is interesting that small molecules, instead of polymers or solvents, can cause such crystal structure transformations. The dissolution of piroxicam at pH 4 is lower than that at pH 1.2. Additionally, the coprecipitates and physical mixtures with C/P mole ratios of 10:1, 20:1, 30:1, 40:1, and 52.5:1 demonstrate substantially higher dissolution of piroxicam compared to that of drug alone.

S-nitrosylation of Bcl-2 inhibits its ubiquitin-proteasomal degradation. A novel antiapoptotic mechanism that suppresses apoptosis.

Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway whose function is dependent on its expression levels. Although Bcl-2 expression is controlled by various mechanisms, post-translational modifications, such as ubiquitination and proteasomal degradation, have emerged as important regulators of Bcl-2 function. However, the underlying mechanisms of this regulation are unclear. We report here that Bcl-2 undergoes S-nitrosylation by endogenous nitric oxide (NO) in response to multiple apoptotic mediators and that this modification inhibits ubiquitin-proteasomal degradation of Bcl-2. Inhibition of NO production by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and by NO synthase inhibitor aminoguanidine effectively inhibited S-nitrosylation of Bcl-2, increased its ubiquitination, and promoted apoptotic cell death induced by chromium (VI). In contrast, the NO donors dipropylenetriamine NONOate and sodium nitroprusside showed opposite effects. The effect of NO on Bcl-2 stability was shown to be independent of its dephosphorylation. Mutational analysis of Bcl-2 further showed that the two cysteine residues of Bcl-2 (Cys158 and Cys229) are important in the S-nitrosylation process and that mutations of these cysteines completely inhibited Bcl-2 S-nitrosylation. Treatment of the cells with other stress inducers, including Fas ligand and buthionine sulfoxide, also induced Bcl-2 S-nitrosylation, suggesting that this is a general phenomenon that regulates Bcl-2 stability and function under various stress conditions. These findings indicate a novel function of NO and its regulation of Bcl-2, which provides a key mechanism for the control of apoptotic cell death and cancer development.