Related Articles

The aim of this study was to develop a temperature-induced polyethylene glycol (PEG) water phase/polysaccharide water-phase emulsion approach for preparing interferon alpha-2b (IFNα-2b)-loaded polysaccharide nanoparticles. IFNα-2b was first added to a mixture of an aqueous solution of PEG and polysaccharide. The mixture solution was stirred in a magnetic stirrer at a rate of 2000 rpm for 45 seconds at 0°C ± 0.5°C. The solution was then prefrozen at different temperatures. The polysaccharide and IFNα-2b partitioned in the polysaccharide phase were preferentially separated out as the dispersed phase from the mixture solution during the prefreezing process. Then the prefrozen sample was freeze-dried to powder form. In order to remove the PEG, the powder was washed with dichloromethane. Once IFNα-2b was loaded into the polysaccharide nanoparticles, these nanoparticles could gain resistance to vapor–water and water–oil interfaces to protect IFNα-2b. The antiviral activity of the polysaccharide nanoparticles in vitro was highly preserved (above 97%), while the antiviral activity of IFNα-2b–loaded polysaccharide nanoparticles using the control water-in-oil-in-water method was only 71%. The antiviral activity of the IFNα-2b from blood samples was also determined on the basis of the activity to inhibit the cytopathic effects of the Sindbis virus on Follicular Lymphoma cells (FL). The antiviral activity in vivo was also highly preserved (above 97%). These polysaccharide nanoparticles could be processed to different formulations according to clinical requirements.

The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum drying, to obtain a better understanding of the effect of protein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing β-lactoglobulin (βLg):mannitol (1∶1–1∶15 wt/wt) were vacuum dried at 5°C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray powder physical state of mannitol was studied using x-ray powder diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1∶5 wt/wt βLg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples except for the 1∶1 wt/wt βLg:mannitol dried sample. FITR studies indicated that a small proportion of crystalline mannitol was present along with the amorphous mannitol in dried samples at lower (less than 1∶5 wt/wt) βLg:mannitol ratios. The Tg of the dried 1∶1 wt/wt βLg:mannitol sample was observed at 33.4°C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the βLg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1∶1 wt/wt in all dried samples. Second-derivative FTTR studies on dried βLg and recombinant human interferon α-2a samples showed that mannitol affected protein secondary structure to a varying degree depending on the overall mannitol content in the dried sample and the type of protein.

The purpose of this study was to investigate the utility of vacuum drying for removing peroxides from polyethylene glycols (PEGs). PEG solutions (PEG 1450 and PEG 20000) containing varying levels of peroxides were prepared by storing under different light and temperature conditions. PEGs containing low and high levels of peroxides were vacuum dried from dilute and concentrated solutions (2.5%, 7.5%, 15%, and 50% wt/vol of PEG 1450 and 2.5%, 7.5%, 15%, and 25% wt/vol of PEG 20000). Ferrous ion oxidation in presence of ferric ion indicator xylenol orange (FOX) colorimetric assay was used to determine the concentration of peroxides. Peroxide content in PEGs increased upon storage. The increase was more pronounced when PEGs were stored at higher temperatures and exposed to light. Vacuum drying at 0.1 mm Hg for 48 hours at 25°C resulted in greater than 90% decrease in the level of peroxides in all cases except when high peroxide containing 25% wt/vol solution of PEG 20000 or 50% wt/vol solution of PEG 1450 were dried. The reduction in the level of peroxides for PEGs dried from high peroxide containing 25% wt/vol solution of PEG 2000 and 50% wt/vol solution of PEG 1450 was found to be 88% and 52%, respectively. Oxidation of methionine in Met-Leu-Phe peptide was significantly reduced when vacuum-dried PEGs were used. Vacuum drying PEG solutions at low pressures is an effective method for the removal of the residual peroxides present in commercially available PEGs.

Novel advanced spray-dried and co-spray-dried inhalable lung surfactant-mimic phospholipid and poly(ethylene glycol) (PEG)ylated lipopolymers as microparticulate/nanoparticulate dry powders of biodegradable biocompatible lipopolymers were rationally formulated via an organic solution advanced spray-drying process in closed mode using various phospholipid formulations and rationally chosen spray-drying pump rates. Ratios of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine PEG (DPPE-PEG) with varying PEG lengths were mixed in a dilute methanol solution. Scanning electron microscopy images showed the smooth, spherical particle morphology of the inhalable particles. The size of the particles was statistically analyzed using the scanning electron micrographs and SigmaScan® software and were determined to be 600 nm to 1.2 μm in diameter, which is optimal for deep-lung alveolar penetration. Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) were performed to analyze solid-state transitions and long-range molecular order, respectively, and allowed for the confirmation of the presence of phospholipid bilayers in the solid state of the particles. The residual water content of the particles was very low, as quantified analytically via Karl Fischer titration. The composition of the particles was confirmed using attenuated total-reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and confocal Raman microscopy (CRM), and chemical imaging confirmed the chemical homogeneity of the particles. The dry powder aerosol dispersion properties were evaluated using the Next Generation Impactor™ (NGI™) coupled with the HandiHaler® dry powder inhaler device, where the mass median aerodynamic diameter from 2.6 to 4.3 μm with excellent aerosol dispersion performance, as exemplified by high values of emitted dose, fine particle fraction, and respirable fraction. Overall, it was determined that the pump rates defined in the spray-drying process had a significant effect on the solid-state particle properties and that a higher pump rate produced the most optimal system. Advanced dry powder inhalers of inhalable lipopolymers for targeted dry powder inhalation delivery were successfully achieved.

The purpose of this research was to develop liposomal dry powder aerosols for protein delivery. The delivery of stable protein formulations is essential for protein subunit vaccine delivery, which requires local delivery to macrophages in the lungs. β-Glucuronidase (GUS) was used as a model protein to evaluate dry powder liposomes as inhaled delivery vehicles. Dimyristoyl phosphatylcholine:cholesterol (7∶3) was selected as the liposome composition. The lyophilization of liposomes, micronization of the powders, aerosolization using a dry powder inhaler (DPI), and in vitro aerodynamic fine particle fraction upon collection in a twinstage liquid impinger were evaluated. After lyophilization and jet-milling, the total amount of GUS and its activity, representing encapsulation efficiency and stability, were evaluated. The GUS amount and activity were measured and compared with freshly-prepared liposomes in the presence of mannitol, 43% of initial GUS amount, 29% of GUS activity after lyophilization and 36% of GUS amount, 22% of activity after micronization were obtained. Emitted doses from dry powder inhaler were 53%, 58%, 66%, and 73% for liposome powder:mannitol carrier ratios of 1∶0, 1∶4, 1∶9, and 1∶19. Fifteen percent of the liposome particles were less than 6.4 μm in aerodynamic diameter. The results demonstrate that milled liposome powders containing protein molecules can be aerosolized effectively at a fixed flow rate. Influences of different cryoprotectants on lyophilization of protein liposome formulations are reported. The feasibility of using liposomal dry powder aerosols for protein delivery has been demonstrated but further optimization is required in the context of specific therapeutic proteins.

Novel nanoparticle-aggregate formulations containing recombinant hepatitis B surface antigen (rHBsAg) were administered to the lungs of guinea pigs and antibodies generated to this antigen evaluated. Preparations of dry powders of: (a) rHBsAg encapsulated within poly(lactic-co-glycolic acid) (PLGA)/polyethylene glycol (PEG) nanoparticles (antigen nanoparticles, AgNSD), (b) rHBsAg in a physical mixture with blank PLGA/PEG nanoparticles (antigen nanoparticle admixture (AgNASD), and (c) rHBsAg encapsulated in PLGA/PEG nanoparticles plus free rHBsAg (antigen nanoparticles and free antigen), were generated by spray drying with leucine. Control groups consisted of alum with adsorbed rHBsAg (AlumAg); reconstituted suspensions of spray-dried rHBsAg-loaded PLGA/PEG nanoparticles with leucine; and rHBsAg-loaded PLGA/PEG nanoparticles (AgN). Control preparations were administered by intramuscular injection; AgN was also spray instilled into the lungs. The IgG titers were measured in the serum for 24 weeks after the initial immunization; IgA titers were measured in the bronchio-alveolar lavage fluid. While the highest titer of serum IgG antibody was observed in guinea pigs immunized with AlumAg administered by the IM route, animals immunized with powder formulations via the pulmonary route exhibited high IgA titers. In addition, guinea pigs immunized with AgNASD via the pulmonary route exhibited IgG titers above 1,000 mIU/ml in the serum (IgG titers above 10 mIU/ml is considered protective). Thus, the disadvantages observed with the existing hepatitis B vaccine administered by the parenteral route may be overcome by administering them as novel dry powders to the lungs. In addition, these powders have the advantage of eliciting a high mucosal immune response in the lungs without traditional adjuvants.

The proteins present in 4% polyethylene glycol (PEG) precipitates of 10 normal sera and 60 samples from patients with rheumatic diseases were studied. A variety of immunochemical methods were used, including estimation of the percentages of total serum proteins precipitated by PEG, gel filtration analyses of the precipitates, and affinity chromatography with protein A and anti-immunoglobulin columns. Substantial amounts of protein were precipitated from normal sera. Many non-immunoglobulin proteins were precipitated from patients' sera, including fibronectin, haptoglobin, albumin, transferrin, and alpha 1-antitrypsin. Affinity chromatography with anti-immunoglobulin columns bound non-immunoglobulin proteins from PEG precipitates, but the protein A affinity column did not do so. The view that circulating antibody-antigen complexes alone are precipitated by 4% PEG is too simplistic; many non-immunoglobulin proteins are involved. They may either bind to immune complexes or be coprecipitated owing to non-specific protein aggregation.

Vacuum foam drying is evaluated as an alternative for lyophilization for enhanced process and storage stability of bovine serum albumin. The protein protective efficiency of different stabilizers was compared in vacuum foam drying and lyophilization. Sucrose mixtures produced better foam characters than mannitol. Unlike calcium lactate, incorporation of polyvinyl pyrrolidone to sucrose synergistically enhanced the recovery of bovine serum albumin. The conformational stability and bovine serum albumin content further increased with sodium phosphate as compared to potassium phosphate. All sucrose mixtures, except calcium lactate showed large α-helix amide-I band at approximately 1656 cm-1. The amorphous powder diffraction in case of sodium phosphate monobasic mixture retained maximum bovine serum albumin content. The crystallization of similar mixtures in lyophilization reduced its bovine serum albumin content. Vacuum foam drying showed better processing and storage stability of bovine serum albumin than lyophilization process. Hence vacuum foam drying is short, simple and industrially economical process for biomolecules preservation.

Background

Molecular Dynamics (MD) simulations are a promising tool to generate molecular understanding of processes related to the purification of proteins. Polyethylene glycols (PEG) of various length are commonly used in the production and purification of proteins. The molecular mechanisms behind PEG driven precipitation, aqueous two-phase formation or the effects of PEGylation are however still poorly understood.

Results

In this paper, we ran MD simulations of single PEG molecules of variable length in explicitly simulated water. The resulting structures are in good agreement with experimentally determined 3D structures of PEG. The increase in surface hydrophobicity of PEG of longer chain length could be explained on an atomic scale. PEG-water interactions as well as aqueous two-phase formation in the presence of PO4 were found to be correlated to PEG surface hydrophobicity.

Conclusions

We were able to show that the taken MD simulation approach is capable of generating both structural data as well as molecule descriptors in agreement with experimental data. Thus, we are confident of having a good in silico representation of PEG.

The preparation, properties, and application in adriamycin delivery of biocompatible and biodegradable poly(lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) nanoparticles are discussed. PLGA-PEG copolymers were synthesized by ring opening polymerization of the dl-lactide and glycolide in the presence of PEG1000.1H-NMR and FT-IR spectrum were consistent with the structure of PLGA-PEG copolymers. The adriamycin-loaded nanoparticles could be prepared using a precipitation-solvent evaporation technique. The nanoparticles have been produced by a precipitation-solvent evaporation technique. The physical characteristics and drug loading efficiency of the PLGA-PEG nanoparticles were influenced by the composition of the PLGA-PEG copolymers used to prepare the nanoparticles. Particle sizes were between 65 and 100 nm for different compositions of PLGA-PEG copolymers. PLGA-PEG nanoparticles prepared from copolymers having relatively high PLGA/PEG ratios were smaller. Entrapment efficiency was 25%–33%. Adriamycin release from the nanoparticles at pH 7.4 showed an initial burst release and then sustained release phase. These results showed that PLGA-PEG nanoparticles could be an effective carrier for cancer therapy.

Background/Aims

Treatment of chronic hepatitis C (CHC) with pegylated interferon α (pegIFNα) and ribavirin results in a sustained response in approximately half of patients. Viral interference with IFNα signal transduction through the Jak-STAT pathway might be an important factor underlying treatment failure. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFNα signaling in cultured cells that express hepatitis C virus (HCV) proteins, and enhance the inhibitory effect of IFNα on HCV replicons. We have performed a clinical study with the aim to evaluate efficacy and safety of the addition of SAMe and betaine to treatment of CHC with pegIFNα/ribavirin.

Methods

In this open-label pilot study, 29 patients with CHC who failed previous therapy with (peg)IFNα/ribavirin were treated with SAMe, betaine, pegIFNα2b and ribavirin. Treatment duration was 6 or 12 months, depending on genotype, and the protocol comprised a stopping rule at week 12 if early virological response (EVR) was not achieved. Virological and biochemical response and safety were assessed throughout the treatment.

Results

29 patients were enrolled and treated according to the study protocol. 79% of the patients were infected with genotype 1, 72% had advanced fibrosis, 76% had previously received pegIFNα/ribavirin, and only 14% achieved EVR to the previous treatment. When treated with the study medications, 17 patients (59%) showed an EVR, only 3 (10%) however achieved a sustained virological response (SVR). SAMe and betaine were found to be safe when used with pegIFNα/ribavirin.

Conclusion

The addition of SAMe and betaine to pegIFNα/ribavirin improves early virological response in CHC.

Trial Registration

ClinicalTrials.gov NCT00310336

Background

Enzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent. The clear understanding of why these preparations show higher catalytic activity than lyophilized powders in organic solvents is not available.

Results

It was found that EPRPs of α-chymotrypsin prepared by precipitation with n-propanol in the presence of trehalose contained substantial amount of trehalose (even though trehalose alone at these lower concentrations was not precipitated by n-propanol). The presence of trehalose in these EPRPs resulted in much higher transesterification rates (45.2 nmoles mg-1min-1) as compared with EPRPs prepared in the absence of trehalose (16.6 nmoles mg-1min-1) in octane. Both kinds of EPRPs gave similar initial transesterification rates in acetonitrile. Use of higher concentrations of trehalose (when trehalose alone also precipitates out), resulted in the formation of PCMCs, which showed higher transesterification rates in both octane and acetonitrile. SEM analysis showed the relative sizes of various preparations. Presence of trehalose resulted in EPRPs of smaller sizes.

Conclusion

The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein. Therefore, the enhancement in the transesterification activity in EPRPs prepared in the presence of trehalose was due to: (a) better retention of essential water layer for catalysis due to the presence of the sugar. This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM. In the case of PCMC, the enhancement in the initial rates was due to an increase in the surface area of the biocatalyst since protein is coated over the core material (trehalose or salt).

It is hoped that the insight gained in this work would help in a better understanding for designing high activity biocatalyst preparation of non-aqueous media.

The aim of this study was to investigate the influence of drying methods and low range of degrees of substitution (DS) on the structural, physicochemical, and drug-release properties of carboxymethyl high-amylose starch (CMS). CMS with three DS of 0.03, 0.14, and 0.25 was synthesized and dried by either solvent precipitation (SP), spray drying (SD), or lyophilization (Ly). DS had an influence on the crystalline structure of CMS. It was found that a DS of 0.14 or higher induced a modification of polymorphism. The drying method and the DS had both an impact on the physical properties of the CMS powder which can further influence the formulation characteristics and drug-release properties from monolithic tablets. The CMS with DS of 0.14 and 0.25 dried by SP or SD presented good excipient properties in terms of compressibility. With acetaminophen (20%) as tracer, the monolithic CMS tablets showed controlled drug release over 17 h for DS of 0.14 and 10 h for DS of 0.25, almost independent of pH, suggesting interesting properties for sustained release applications.

Background

Interferon alpha (IFNα) therapy has been widely used in the treatment of chronic hepatitis B (CHB) for decades. Nucleos(t)ide analogues are also increasingly used to treat CHB recently. More and more studies are being carried out concerning the clearance or seroconversion of HBsAg, which is recognized as an ideal goal of CHB therapy. This study conducted a meta-analysis to estimate the effect of pegylated interferon alpha (peginterferon α, PEG-IFNα)-based therapy on HBsAg clearance or seroconversion in CHB.

Methods

All available controlled clinical trials, published from 2004 to 2010, with the following antiviral therapies for CHB patients: PEG-IFNα combined with lamivudine (LAM), PEG-IFNα only, conventional IFNα and LAM, with a course ≥24 weeks, were meta-analysed for HBsAg clearance and seroconversion.

Results

Fourteen trials (involving a total of 2,682 patients) were identified, including seven high-quality and seven low-quality studies. The analysis results of the different antiviral therapies on HBsAg clearance or seroconversion were as follows: 1. No significant difference in HBsAg clearance or seroconversion was observed between the combination therapy group and PEG-IFNα monotherapy group [odds ratio (OR) = 1.16, 95% confidence intervals (CI) (0.73-1.85), P = 0.54 and OR = 1.07, 95% CI (0.58-1.97), P = 0.82, respectively]; 2. HBsAg clearance and seroconversion rates in patients with combination therapy were markedly higher than in those with LAM monotherapy [OR = 9.41, 95% CI (1.18-74.94), P = 0.03, and OR = 12.37, 95% CI (1.60-95.44), P = 0.02, respectively]; 3. There was significant difference in HBsAg clearance between the PEG-IFNα group and IFNα monotherapy group [OR = 4.95, 95% CI (1.23-20.00), P = 0.02], but not in seroconversion [OR = 2.44, 95% CI (0.35-17.08), P = 0.37]; 4. PEG-IFNα was superior to LAM in HBsAg seroconversion [OR = 14.59, 95% CI (1.91-111.49), P = 0.01].

Conclusions

PEG-IFNα facilitated HBsAg clearance or seroconversion in CHB patients. PEG-IFNα-based therapy was more effective than LAM monotherapy in achieving HBsAg clearance or seroconversion for both HBeAg-positive and HBeAg-negative CHB patients. There was no significant difference in HBsAg clearance or seroconversion between PEG-IFNα/LAM combination therapy and PEG-IFNα monotherapy. PEG-IFNα was obviously superior to conventional IFNα in HBsAg clearance, but not in HBsAg seroconversion. Although PEG-IFNα produced significantly higher rates of HBsAg clearance and seroconversion, the absolute change in the proportion of HBsAg clearance and seroconversion was low (about 3-6%). Therefore, additional interventions are needed to improve the rate of positive outcomes.

Polyethylene glycol-phospholipid micelles form a major class of nanocarriers in pharmacy and medicine due to proven capability in drug solubilization, sustained drug release, and evidence for targeted drug delivery in vivo. In this report, we have prepared micelles composed of PEG-block-poly(N-hexyl stearate L-aspartamide) (PEG-b-PHSA), having nine stearic acid side chains and have studied their stability in the presence of serum proteins by Förster resonance energy transfer (FRET) experiments. In the presence of serum albumin, alpha and beta globulins, or gamma globulins, there are minimal changes in FRET over two hours in vitro, indicating integrity of PEG-b-PHSA micelles. In contrast, 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethylene glycol)-5000] (PEG-DSPE) micelles lose FRET over two hours in vitro, especially in the presence of alpha and beta globulins, indicating the disruption of PEG-DSPE micelles and leakage of fluorescent probes. Owing to the aliphatic nature of DSPE and PHSA, both PEG-b-PHSA and PEG-DSPE micelles efficiently solubilize amphotericin B (AmB), a poorly water-soluble antifungal agent used to combat systemic mycoses. However, only PEG-b-PHSA micelles gradually liberate AmB in the presence of alpha and beta globulins, based on time-dependent changes in self-aggregation state of AmB, monitored by UV/VIS spectroscopy. PEG-b-PHSA micelles are remarkably stable in the presence of serum proteins and a more stable alternative for poorly water soluble drugs, which have been solubilized by PEG-DSPE micelles.

The mechanism of the interferon-alpha (IFNα)-induced antiviral response is not completely understood. We recently examined the transcriptional response to IFNα in uninfected chimpanzees. The transcriptional response to IFNα in the liver and peripheral blood mononuclear cells (PBMC) was rapidly induced but was also rapidly down-regulated, with most IFNα stimulated genes (ISGs) returning to baseline within 24 hr. We have extended these observations to include chimpanzees chronically infected with hepatitis C virus (HCV). Remarkably, using total genome microarray analysis, almost no induction of ISG transcripts was observed in the liver of chronically infected animals following IFNα dosing, while the response in PBMC was similar to that in uninfected animals. Consistent with this finding, no decrease in viral load occurred in up to 12 weeks of pegylated (peg)-IFNα therapy. The block in response to exogenous IFNα appeared to be HCV specific, since the response in an HBV infected animal was similar to that of uninfected animals. The lack of response to exogenous IFNα may be due to an already maximally induced ISG response, since HCV chronically infected chimpanzees already have a highly up-regulated hepatic ISG response. Alternatively, negative regulation may block the response to exogenous IFNα, yet does not prevent the continued response to endogenous ISG stimuli. The IFNα response in HCV chronically infected chimpanzees may be mechanistically similar to the null response in the human population.

Conclusion

In chimpanzees infected with HCV, the highly elevated hepatic ISG expression may prevent further induction of ISGs and antiviral efficacy following IFNα treatment.

The influence of cholesterol (Chol) in the liposomal bilayer on the properties of inhalable protein-loaded liposomal powders prepared by spray-drying technique was investigated. Lysozyme (LSZ) was used as a model protein. Feed solution for spray drying was prepared by direct mixing of aqueous solution of LSZ with mannitol solution and empty liposome dispersions composed of hydrogenated phosphatidylcholine and Chol at various molar ratios. The spray-dried powders were characterized with respect to morphology, thermal property, and crystallinity using scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction, respectively. Most formulations gave slightly aggregated, spherical particles, and percentage yields of the spray-dried powders decreased with increasing Chol content. Degree of particle aggregation depended on the powder composition. The powders spontaneously formed liposomes which efficiently entrapped LSZ after reconstitution with HEPES buffered saline (HBS) at 37°C. Lysozyme entrapment efficiency and size distribution of the reconstituted liposomes were evaluated after the powders were reconstituted with HBS. Increasing Chol content resulted in a decrease in size of the reconstituted liposomes and an increase in entrapment efficiency of LSZ. These results correlated with thermal behaviors of the reconstituted liposomes. Biological activity of LSZ was not affected by the spray-drying process. It was also demonstrated that LSZ-loaded liposomal powders could be produced without the need to preload the LSZ into liposomes prior to spray-drying process.

Here we report the stabilization of the nitric oxide (NO) prodrugs and anti-cancer lead compounds, PABA/NO (O2-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate) and “Double JS-K” (1,5-bis{[1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-2-ato]-2,4-dinitrobenzene), through their incorporation into polymer-protected nanoparticles. The prodrugs were formulated in block copolymer-stabilized nanoparticles with sizes from 220 to 450 nm by a novel rapid precipitation process. The block copolymers, with polyethylene glycol (PEG) soluble blocks, provide a steric barrier against NO prodrug activation by glutathione. Too rapid activation and NO release has been a major barrier to effective administration of this class of compounds. The nanoparticle stabilized PABA/NO from attack by glutathione as evidenced by a significant increase in time taken for 50% decomposition from 15 min (unformulated) to 5 h (formulated); in the case of Double JS-K, the 50% decomposition time was extended from 4.5 min (unformulated) to 40 min (formulated). The more hydrophobic PABA/NO produced more stable nanoparticles and correspondingly more extended release times in comparison with Double JS-K. The hydrophobic blocks of the polymer were either polystyrene or polylactide. Both blocks produced nanoparticles of approximately the same size and release kinetics. This combination of PEG-protected nanoparticles with sizes appropriate for cancer targeting by enhanced permeation and retention (EPR) and delayed release of NO may afford enhanced therapeutic benefit.

The purpose of this research was to apply vacuum foam drying (VFD) for processing of LaSota virus and to screen formulation additives for its stability. The aqueous dispersion of harvest containing sucrose or trehalose in combination with additive (monosaccharides, polymers, N-Z-amine) was prepared. The diluted dispersions in vials were vacuum concentrated, foamed to form a continuous structure, and vacuum dried. The products were evaluated for foam characteristics, residual moisture, virus titer, x-ray diffraction pattern, and stability profile. The foamability increased with solid content in solutions. The foamability of sucrose was enhanced with incorporation of N-Z-amine (10% and 15% wt/vol) and polyvinyl pyrrolidone (PVP K30, 3% wt/vol). The fructose- or galactose-containing mixtures were deposited irregularly on the vial surface. The virus titer increased with disaccharides in the formulation. Sucrose provided better protection than trehalose. Unlike lyophilization, N-Z-amine with sucrose protected the virus from Millard’s Browning. Amino acids do not have a catalytic effect on hydrolysis of sucrose during VFD. Monosaccharides were ineffective. A synergistic effect of PVP K30 or polyethylene glycol 6000 (3% wt/vol) with N-Z-amine provided the maximum virus titer (6.97 and 7.15, respectively). This formulation retained the desired virus potency at 5°, 25°, and 40°C. The diffraction pattern revealed that a threshold concentration of N-Z-amine was required for inhibiting crystallization of sucrose during VFD. VFD was successfully applied to produce a solid LaSota formulation. The products were amorphous and did not devitrify on storage.

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)

A nephritic condition was developed by infecting Swiss Webster albino mice with the malarial parasite Plasmodium berghei NK 65. These animals were tested for urinary protein and the presence of circulating immune complexes using reagent strips and a polyethylene glycol (PEG) precipitation assay. The circulating immune complexes were isolated from the sera using both affinity chromatography and PEG precipitation and from the kidney by acid elution. The isolated complexes were dissociated into their individual components and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The components of the complexes were transferred to nitrocellulose sheets and probed for the presence of malarial antigens using a rabbit anti-P berghei antisera. The overall humoral response to the malarial parasite was evaluated using a radial immunodiffusion assay. The present study confirmed that the malarial-infected animals not only developed the nephritic condition (as evident by the high levels of proteinuria) but also, as indicated by the PEG assay, have the presence of high levels of circulating immune complexes in their serum. The apparent absence in the SDS gels of any abnormal protein bands followed by the inability of the Western blot to reveal any malarial antigens provides some of the strongest evidence to date that these malarial proteins are not directly involved in the circulating immune complexes believed to be responsible for producing this nephritic condition.

Images

We formulated PA-824, a nitroimidazopyran with promise for the treatment of tuberculosis, for efficient aerosol delivery to the lungs in a dry powder porous particle form. The objectives of this study were to prepare and characterize a particulate form of PA-824, assess the stability of this aerosol formulation under different environmental conditions, and determine the pharmacokinetic parameters for the powder after pulmonary administration. The drug was spray dried into porous particles containing a high drug load and possessing desirable aerosol properties for efficient deposition in the lungs. The physical, aerodynamic, and chemical properties of the dry powder were stable at room temperature for 6 months and under refrigerated conditions for at least 1 year. Pharmacokinetic parameters were determined in guinea pigs after the pulmonary administration of the PA-824 powder formulation at three doses (20, 40, and 60 mg/kg of body weight) and compared to those after the intravenous (20 mg/kg) and oral (40 mg/kg) delivery of the drug. Oral and inhaled delivery of PA-824 achieved equivalent systemic delivery at the same body dose within the first 12 h of dosing. However, animals dosed by the pulmonary route showed drug loads that remained locally in the lungs for 32 h postexposure, whereas those given the drug orally cleared the drug more rapidly. Therefore, we expect from these pharmacokinetic data that pulmonary delivery may achieve the same efficacy as oral delivery at the same body dose, with a potential improvement in efficacy related to pulmonary infection. This may translate into the ability to deliver lower body doses of this drug for the treatment of tuberculosis by aerosol.

Background and the purpose of the study

Because of its intense bitter taste and susceptibility to moisture Cefetamet Pivoxil (CPH) is presently available only in the form of tablet. The aim of this study was to develop taste masked CPH dry powder suspension.

Methods

Methods employed for formulations were: a) Film coating of CPH using Eudragit E100 and subsequent adsorption on different carriers such as spray-dried lactose, sodium starch glycolate and spray-dried mannitol and b) Complexation of CPH with three different ion exchange resins indion 234 amberlite IRP64 and amberlite IRP69.

Results

Taste viz evaluation as recognized by volunteers revealed that coating with Eudragit E100 and subsequent adsorption on different carriers do not mask the bitter taste of the drug. Suspensions prepared using amberlite IRP64 and amberlite IRP69 were extremely palatable with no bitter after taste. They showed pseudoplastic flow behavior and were too viscous even after shearing for sufficient duration of time and exhibited poor pourability. The suspension made with indion 234 was palatable with slight or no bitter after taste. It demonstrated plastic flow with negligible thixotropy. It had moderate viscosity at rest and could be poured after a reasonable amount of shaking. CPH dry powder suspensions were very unstable under different conditions except under refrigeration. A 5% degradation of drug was occurred in reconstituted suspension in 4 days period when stored at room temperature.

Conclusion

Dry powder suspension prepared with indion 234 having 5% overages was stable even after 4th day of reconstitution and palatable with slight or no bitter after taste.

This article reports the development of a method to purify and concentrate intact virions from oyster extracts to volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase PCR (RT-PCR) and confirmation by oligonucleotide probe hybridization. Fifty-gram oyster samples were processed by an adsorption-elution -precipitation method and then seeded with 10(1) to 10(5) PFU of poliovirus type 1 (PV1) or hepatitis A virus (HAV). Seeded viruses in oyster extracts were purified by fluorocarbon extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Virus recovery after elution of PEG precipitates was dependent upon PEG concentration and averaged 60% for PV1 and 40% for HAV. The next processing step used the protein-precipitating agent Pro-Cipitate (Affinity Technology, Inc., Brunswick, N.J.) in an adsorption-elution -precipitation scheme to further concentrate viruses and reduce sample volumes to 100 microliter. Oyster extracts processed by Pro-Cipitate adsorption-elution-precipitation were directly compatible with RT-PCR and yielded virus recoveries of > 80% for both PV1 and HAV. When extracts from 50-g oyster samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 10 PFU for both PV1 and HAV and with low levels of Norwalk virus. Virus recoveries based on cell culture infectivity were 25 to 35% for PV1 and 5 to 10% for HAV. When tested on artificially contaminated raw oysters, the combined method successfully detected > or = 10(3) PFU of PV1 and HAV and 10(5) RT-PCR-amplifiable units of Norwalk virus. Virus detection by RT-PCR and cell culture infectivity was consistent and well correlated among replicate samples and at different virus titers. The procedure developed in this study is rapid, sensitive, and effective for the direct detection of enteric viruses in oysters by RT-PCR.

Objective(s)

The aim of this study was to investigate the possibility of production of ibuprofen pellets with high amount of rate retarding polymer by aid of PEG400 as plasticizer.

Materials and Methods

Polyethylene glycol (PEG400) in concentrations of 1, 3 or 5% w/w with respect to Eudragit RL was used in production of pellets containing 60% ibuprofen and 40% excipient (2% polyvinylpyrrolidone (PVP), 7.6 or 0% microcrystalline cellulose (MCC) and 30.4 or 38% Eudragit RL). Physicomechanical and release properties of pellets were evaluated.

Results

In presence of PEG400, formulations containing 30.4% Eudragit RL and 7.6% MCC could easily form pellets. In formulations without any MCC pellets were obtained only in presence of 3 or 5% PEG400. Pellets containing MCC with 0 or 1% PEG400 showed brittle properties but those with 3% or 5% PEG400 showed plastic nature under pressure. Elastic modulus dramatically decreased with increasing PEG400 indicating softening of pellets. This was due to shift of Eudragit structure from glassy to rubbery state which was supported by DSC studies. Mean dissolution time (MDT) increased with addition of 1 or 3% PEG400 but this was not the case for pellets with 5% PEG400.

Conclusion

Overall PEG400 is a potential plasticizer in production of pellets based on Eudragit RL and ibuprofen. The ease in process of extrusion-spheronization, increasing the mean dissolution time and change in mechanical properties of pellets from brittle to plastic behavior were advantages of using PEG400. Changes in mechanical properties of pellets are important when pellets are intended to be compressed as tablets.