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1.  Effect of vacuum drying on protein-mannitol interactions: The physical state of mannitol and protein structure in the dried state 
AAPS PharmSciTech  2004;5(1):58-69.
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.
doi:10.1208/pt050110
PMCID: PMC2784861  PMID: 15198531
mannitol; proteins; vacuum drying; amorphous; protein structure
2.  Preparation of bioactive interferon alpha–loaded polysaccharide nanoparticles using a new approach of temperature-induced water phase/water-phase emulsion 
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.
doi:10.2147/IJN.S35502
PMCID: PMC3439862  PMID: 22973103
activity of interferon alpha-2b; interferon alpha-2b; stability of interferon alpha-2b; dextran; nanoparticles
3.  Vacuum foam drying for preservation of LaSota virus: Effect of additives 
AAPS PharmSciTech  2006;7(3):E30-E37.
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.
doi:10.1208/pt070360
PMCID: PMC2750502  PMID: 17025241
Foam drying; LaSota virus; additives; stabilization
4.  Removal of peroxides in polyethylene glycols by vacuum drying: Implications in the stability of biotech and pharmaceutical formulations 
AAPS PharmSciTech  2006;7(3):E47-E53.
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.
doi:10.1208/pt070362
PMCID: PMC2750504  PMID: 16796364
Freeze drying; peroxides; polyethylene glycol; proteins; vacuum drying
5.  Vacuum Foam Drying: An Alternative to Lyophilization for Biomolecule Preservation 
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.
doi:10.4103/0250-474X.103837
PMCID: PMC3546342  PMID: 23325988
Bovine serum albumin; inorganic phosphates; lyophilization; saccharides; vacuum foam drying
6.  The Effects of Excipients and Particle Engineering on the Biophysical Stability and Aerosol Performance of Parathyroid Hormone (1-34) Prepared as a Dry Powder for Inhalation 
AAPS PharmSciTech  2011;12(1):304-311.
Pulmonary delivery of therapeutic peptides and proteins has many advantages including high relative bioavailability, rapid systemic absorption and onset of action and a non-invasive mode of administration which improves patient compliance. In this study, we investigated the effect of spray-drying (SD) and spray freeze-drying processes on the stability and aerosol performance of parathyroid hormone (PTH) (1-34) microparticles. In this study, the stabilisation effect of trehalose (a non-reducing sugar) and Brij 97 (a non-ionic surfactant) on spray-dried PTH particles was assessed using analytical techniques including circular dichroism (CD), fluorescence spectroscopy, modulated differential scanning calorimetry and an in vitro bioactivity assay. Physical characterisation also included electron microscopy, tap density measurement and laser light diffraction. The aerosol aerodynamic performance of the formulations was assessed using the Andersen cascade impactor. Based on these studies, a formulation for spray freeze-drying was selected and the effects of the two particle engineering techniques on the biophysical stability and aerosol performance of the resulting powders was determined. CD, fluorescence spectroscopy and bioactivity data suggest that trehalose when used alone as a stabilising excipient produces a superior stabilising effect than when used in combination with a non-ionic surfactant. This highlights the utility of CD and fluorescence spectroscopy studies for the prediction of protein bioactivity post-processing. Therefore, a method and formulation suitable for the preparation of PTH as a dry powder was developed based on spray-drying PTH with trehalose as a stabiliser with the bioactivity of SD PTH containing trehalose being equivalent to that of unprocessed PTH.
doi:10.1208/s12249-011-9585-2
PMCID: PMC3066375  PMID: 21271316
parathyroid hormone; pulmonary delivery; spray drying; spray freeze-drying; stability
7.  New inhalation-optimized itraconazole nanoparticle-based dry powders for the treatment of invasive pulmonary aspergillosis 
Purpose
Itraconazole (ITZ) dry powders for inhalation (DPI) composed of nanoparticles (NP) embedded in carrier microparticles were prepared and characterized.
Methods
DPIs were initially produced by reducing the ITZ particle size to the nanometer range using high-pressure homogenization with tocopherol polyethylene 1000 succinate (TPGS, 10% w/w ITZ) as a stabilizer. The optimized nanosuspension and the initial microsuspension were then spray-dried with different proportions of or in the absence of mannitol and/or sodium taurocholate. DPI characterization was performed using scanning electron microscopy for morphology, laser diffraction to evaluate the size-reduction process, and the size of the dried NP when reconstituted in aqueous media, impaction studies using a multistage liquid impactor to determine the aerodynamic performance and fine-particle fraction that is theoretically able to reach the lung, and dissolution studies to determine the solubility of ITZ.
Results
Scanning electron microscopy micrographs showed that the DPI particles were composed of mannitol microparticles with embedded nano- or micro-ITZ crystals. The formulations prepared from the nanosuspension exhibited good flow properties and better fine-particle fractions, ranging from 46.2% ± 0.5% to 63.2% ± 1.7% compared to the 23.1% ± 0.3% that was observed with the formulation produced from the initial microsuspension. Spray-drying affected the NP size by inducing irreversible aggregation, which was able to be minimized by the addition of mannitol and sodium taurocholate before the drying procedure. The ITZ NP-based DPI considerably increased the ITZ solubility (58 ± 2 increased to 96 ± 1 ng/mL) compared with that of raw ITZ or an ITZ microparticle-based DPI (<10 ng/mL).
Conclusion
Embedding ITZ NP in inhalable microparticles is a very effective method to produce DPI formulations with optimal aerodynamic properties and enhanced ITZ solubility. These formulations could be applied to other poorly water-soluble drugs and could be a very effective alternative for treating invasive pulmonary aspergillosis.
doi:10.2147/IJN.S34091
PMCID: PMC3477927  PMID: 23093903
aspergillosis; spray-drying; homogenization; inhalation; saturation; solubility
8.  Liposomal dry powders as aerosols for pulmonary delivery of proteins 
AAPS PharmSciTech  2005;6(4):E641-E648.
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.
doi:10.1208/pt060480
PMCID: PMC2750613  PMID: 16408866
protein; liposome; lyophilization; dry powders; aerosol; pulmonary delivery
9.  Peginterferon alpha-based therapy for chronic hepatitis B focusing on HBsAg clearance or seroconversion: a meta-analysis of controlled clinical trials 
BMC Infectious Diseases  2011;11:165.
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.
doi:10.1186/1471-2334-11-165
PMCID: PMC3128052  PMID: 21651820
hepatitis B; HBsAg; peginterferon; interferon; lamivudine
10.  Preparation and in vivo absorption evaluation of spray dried powders containing salmon calcitonin loaded chitosan nanoparticles for pulmonary delivery 
Purpose
The aim of the present study was to prepare inhalable co-spray dried powders of salmon calcitonin loaded chitosan nanoparticles (sCT-CS-NPs) with mannitol and investigate pulmonary absorption in rats.
Methods
The sCT-CS-NPs were prepared by the ionic gelation method using sodium tripolyphosphate (TPP) as a cross-linking polyion. Inhalable dry powders were obtained by co-spray drying aqueous dispersion of sCT-CS-NPs and mannitol. sCT-CS-NPs co-spray dried powders were characterized with respect to morphology, particle size, powder density, aerodynamic diameter, protein integrity, in vitro release of sCT, and aerosolization. The plasmatic sCT levels following intratracheal administration of sCT-CS-NPs spray dried powders to the rats was also determined.
Results
sCT-CS-NPs were able to be incorporated into mannitol forming inhalable microparticles by the spray drying process. The sCT-CS-NPs/mannitol ratios and spray drying process affected the properties of the microparticles obtained. The conformation of the secondary structures of sCTs was affected by both mannitol content and spray dry inlet temperature. The sCT-CS-NPs were recovered after reconstitution of spray dried powders in an aqueous medium. The sCT release profile from spray dried powders was similar to that from sCT-CS-NPs. In vitro inhalation parameters measured by the Andersen cascade impactor indicated sCT-CS-NPs spray dried powders having promising aerodynamic properties for deposition in the deep lung. Determination of the plasmatic sCT levels following intratracheal administration to rats revealed that the inhalable sCT-CS NPs spray dried powders provided higher protein absorption compared to native sCT powders.
Conclusion
The sCT-CS-NPs with mannitol based spray dried powders were prepared to have appropriate aerodynamic properties for pulmonary delivery. The developed system was able to deliver sCT via a pulmonary route into the systemic circulation.
doi:10.2147/DDDT.S47681
PMCID: PMC3770519  PMID: 24039397
Salmon calcitonin; chitosan; nanoparticles; mannitol; spray dried powders; pulmonary delivery
11.  Immunization of Guinea Pigs with Novel Hepatitis B Antigen as Nanoparticle Aggregate Powders Administered by the Pulmonary Route 
The AAPS Journal  2010;12(3):330-337.
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.
doi:10.1208/s12248-010-9192-2
PMCID: PMC2895445  PMID: 20419360
antibody titer; dry powder formulation; hepatitis B vaccine; pulmonary delivery
12.  Design, physicochemical characterization, and optimization of organic solution advanced spray-dried inhalable dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine poly(ethylene glycol) (DPPE-PEG) microparticles and nanoparticles for targeted respiratory nanomedicine delivery as dry powder inhalation aerosols 
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.
doi:10.2147/IJN.S30724
PMCID: PMC3552552  PMID: 23355776
biocompatible biodegradable lipopolymers; lung surfactant; pulmonary delivery; self-assemblies; solid-state; lipospheres
13.  Preservation of the Immunogenicity of Dry-powder Influenza H5N1 Whole Inactivated Virus Vaccine at Elevated Storage Temperatures 
The AAPS Journal  2010;12(2):215-222.
Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20°C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40°C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40°C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40°C may also point to an extended shelf-life when stored at 4°C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.
doi:10.1208/s12248-010-9179-z
PMCID: PMC2844510  PMID: 20195930
freeze-drying; inulin; pandemic influenza; vaccine stockpiling; whole inactivated influenza vaccine (H5N1)
14.  Preservation of the Immunogenicity of Dry-powder Influenza H5N1 Whole Inactivated Virus Vaccine at Elevated Storage Temperatures 
The AAPS Journal  2010;12(2):215-222.
Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20°C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40°C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40°C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40°C may also point to an extended shelf-life when stored at 4°C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.
doi:10.1208/s12248-010-9179-z
PMCID: PMC2844510  PMID: 20195930
freeze-drying; inulin; pandemic influenza; vaccine stockpiling; whole inactivated influenza vaccine (H5N1)
15.  Development of a bladder instillation of the indoloquinone anticancer agent EO-9 using tert-butyl alcohol as lyophilization vehicle 
AAPS PharmSciTech  2007;8(3):E78-E87.
The purpose of this research was to develop a stable bladder instillation of EO-9 for the treatment of superficial bladder cancer. First, stability and dissolution studies were performed. Subsequently, the freeze-drying process was optimized by determination of the freeze-drying characteristics of the selected cosolvent/water system and differential scanning calorimetry analysis of the formulation solution. Furthermore, the influence of the freeze-drying process on crystallinity and morphology of the freeze-dried product was determined with x-ray diffraction analysis and scanning electron microscopy, respectively. Subsequently, a reconstitution solution was developed. This study revealed that tert-butyl alcohol (TBA) can be used to both dramatically improve the solubility and stability of EO-9 and to shorten the freeze-drying cycle by increasing the sublimation rate. During freeze drying, 3 TBA crystals were found: TBA hydrate-ice crystals, crystals of TBA hydrate, and a third crystal, probably composed of TBA hydrate crystals containing ≈90% to 95% TBA. Furthermore, it was shown that crystallization of TBA hydrate was inhibited in the presence of both sodium bicarbonate (NaHCO3) and mannitol. Addition of an annealing step resulted in a minor increase in the crystallinity of the freeze-dried product and formation of the δ-polymorph of mannitol. A stable bladder instillation was obtained after reconstitution of the freeze-dried product (containing 8 mg of EO-9, 20 mg of NaHCO3, and 50 mg of mannitol per vial) to 20 mL with a reconstitution solution composed of propylene glycol/water for injection (WfI)/NaHCO3/sodium edetate 60%/40%/2%/0.02% vol/vol/wt/wt, followed by dilution with Wfl to a final volume of 40 mL.
doi:10.1208/pt0803061
PMCID: PMC2750557  PMID: 17915828
Mitomycin analogue; EO-9; formulation development; freeze drying; tert-butyl alcohol
16.  The protective effect of lactose on lyophilization of CNK-20402 
AAPS PharmSciTech  2005;6(1):E42-E48.
The goal of this research was to assess the feasibility of using lyophilization to stabilize an exploratory compound, CNK-20402, with a minimal amount of impurity (CNK-20193) formation. A mixed-level full factorial experimental design was used to screen excipients of glycine, mannitol, lactose monohydrate, and povidone K-12. Cryostage microscopy, powder X-ray diffraction, Karl Fischer titration, HPLC, and water vapor sorption were used to assess the formulations' physicochemical properties and stability. Initial physical characterization from powder X-ray diffraction revealed that the mannitol- and glycine-containing formulations were crystalline with the patterns of the pure excipient, whereas the remaining formulations were amorphous in structure. Chemically, the formulations stored at 50°C for 1 month had 2.36%, 1.05%, 0.81%, 0.79%, and 0.49% CNK-20193 for glycine, mannitol, drug alone, povidone K-12, and lactose formulations, respectively. The formulations containing drug-mannitol, drug alone, and druglactose were selected for accelerated stability study based on statistical analysis. Recovery of CNK-20193 in these formulations was 1.22%, 1.00%, and 0.55%, respectively, when stored at 40°C/75% relative humidity storage conditions for 3 months. Water vapor sorption analysis revealed weight gains of over 7%, 21%, and 24% for the mannitol, lactose, and drug alone formulations, respectively. Testing formulations with different concentrations of lactose by water vapor sorption indicated that CNK-20402 concentrations as low as 10% (wt/wt) could inhibit the recrystallization of lactose. The lactose-containing formulation exhibited the best stability among the formulations tested. The protective mechanism of lactose on the CNK-20402, based on water vapor sorption studies, is believed to be a result of (1) the drug-lactose interaction, and (2) competition between lactose and drug for the residual water in the formulation.
doi:10.1208/pt060109
PMCID: PMC2750410  PMID: 16353962
solubility; stability; lactose; mannitol; lyophilization; water vapor sorption
17.  Agglomerates Containing Pantoprazole Microparticles: Modulating the Drug Release 
AAPS PharmSciTech  2009;10(2):335-345.
Pantoprazole-loaded microparticles were prepared using a blend of Eudragit® S100 and Methocel® F4M. The accelerated stability was carried out during 6 months at 40°C and 75% relative humidity. In order to improve technological characteristics of the pantoprazole-loaded microparticles, soft agglomerates were prepared viewing an oral delayed release and gastro-resistant solid dosage form. The agglomeration was performed by mixing the pantoprazole microparticles with spray-dried mannitol/lecithin powders. The effects of factors such as the amount of lecithin in the spray-dried mannitol/lecithin powders and the ratio between pantoprazole microparticles and spray-dried mannitol/lecithin powders were evaluated. The pantoprazole-loaded microparticles present no significant degradation in 6 months. The agglomerates presented spherical shape, with smooth surface and very small quantity of non-agglomerated particles. The agglomerates presented different yields (35.5–79.0%), drug loading (58–101%), and mechanical properties (tensile strength varied from 44 to 69 mN mm−2), when the spray-dried mannitol/lecithin powders with different lecithin amounts were used. The biopharmaceutical characteristics of pantoprazole microparticles, i.e., their delayed-release properties, were not affected by the agglomeration process. The gastro-resistance of the agglomerates was affected by the amount of spray-dried mannitol/lecithin powders. The ratio of lecithin in the spray-dried mannitol/lecithin powders was the key factor in the agglomerate formation and in the drug release profiles. The agglomerates presenting better mechanical and biopharmaceutical characteristics were prepared with 1:2 (w/w) ratio of pantoprazole-loaded microparticles and mannitol/lecithin (80:20) powder.
doi:10.1208/s12249-009-9214-5
PMCID: PMC2690777  PMID: 19319687
agglomerates; delayed release; gastro-resistance; microparticles
18.  Development and characterization of dilutable self-microemulsifying premicroemulsion systems (SMEPMS) as templates for preparation of nanosized particulates 
The utilization of self-microemulsifying premicroemulsion systems (SMEPMS) as templates for preparing poorly water-soluble compounds in the nanosized range represents a promising strategy. Fenofibrate was formulated with n-butyl L-lactate, Tween 80, and a number of cosurfactants (ethanol, 1-propanol, and PEG 600), diluted with the water phase (either water or saccharide solution) and then subjected to a freeze-drying (FD) process to obtain SMEPMS nanosized particulates. Results demonstrated that the particle size after resuspension of these FD SMEPMS nanosized particulates in water was too large, so the addition of saccharide solutions (lactose, mannitol, glucose, sucrose, and trehalose) as the solid carrier to prevent particles from aggregating seemed to be necessary and workable due to steric hindrance and repulsion. However, instability of these resuspended FD nanosized particulates after 30–90 minutes still occurred, and the addition of 0.5% sodium lauryl sulfate in the resuspending medium was able to retard the aggregation and maintain the particle size within the nano-range. Evaluation by scanning electron microscopy and X-ray powder diffraction also confirmed the results. It was concluded that using an SMEPMS formulation with PEG 600 as the cosurfactant, and in the presence of a suitable saccharide as an anticaking agent and FD process were able to produce fenofibrate nanoparticles.
doi:10.2147/IJN.S48465
PMCID: PMC3775676  PMID: 24049445
fenofibrate; saccharides; freeze-drying; nanoparticles
19.  Study on the Effect of Solution Conditions on Heat Induced-Aggregation of Human Alpha Interferon  
A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b (rhIFNα2b) has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFNα2b. The protein was incubated for 1 h at 40–70 °C and for up to 240 h at 50 °C and its aggregation tendency was then studied using optical density (at 350 nm), SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH (5, 6 and 7) and buffer concentrations (10, 55 and 100 mM) on the aggregation of protein following incubation at 50 °C for 72 h was also evaluated. The results obtained for samples incubated at 50 °C for up to 240 h showed that OD350 and the amount of higher molecular weight aggregates (HMW) increased and the monomer content decreased significantly (p<0.05) as the incubation time increased. Following incubation at various temperatures, a significant increase in OD350, drop in monomer content and increase in the amount of HMW aggregates were observed (p<0.05). Data obtained from incubation of samples at 50 °C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 (p<0.05). At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFNα2b.
PMCID: PMC3977050  PMID: 24711826
Interferon alpha2b; Thermal aggregation; SE-HPLC; Optical density; SDS-PAGE
20.  Stability evaluation of freeze-dried Lactobacillus paracasei subsp. tolerance and Lactobacillus delbrueckii subsp. bulgaricus in oral capsules 
Freeze-drying is a common preservation technology in the pharmaceutical industry. Various studies have investigated the effect of different cryoprotectants on probiotics during freeze-drying. However, information on the effect of cryoprotectants on the stability of some Lactobacillus strains during freeze-drying seems scarce. Therefore, the aim of the present study was to establish production methods for preparation of oral capsule probiotics containing Lactobacillus paracasei subsp. tolerance and Lactobacillus delbrueckii subsp. Bulgaricus. It was also of interest to examine the effect of various formulations of cryoprotectant media containing skim milk, trehalose and sodium ascorbate on the survival rate of probiotic bacteria during freeze-drying at various storage temperatures. Without any cryoprotectant, few numbers of microorganisms survived. However, microorganisms tested maintained higher viability after freeze-drying in media containing at least one of the cryoprotectants. Use of skim milk in water resulted in an increased viability after lyophilization. Media with a combination of trehalose and skim milk maintained a higher percentage of live microorganisms, up to 82%. In general, bacteria retained a higher number of viable cells in capsules containing freeze-dried bacteria with sodium ascorbate after three months of storage. After this period, a marked decline was observed in all samples stored at 23°C compared to those stored at 4°C. The maximum survival rate (about 72-76%) was observed with media containing 6% skim milk, 8% trehalose and 4% sodium ascorbate.
PMCID: PMC3500555  PMID: 23181077
Probiotics; Lactobacillus delbrueckii; Lactobacillus paracasei; Freeze-drying
21.  S-Adenosyl-Methionine and Betaine Improve Early Virological Response in Chronic Hepatitis C Patients with Previous Nonresponse 
PLoS ONE  2010;5(11):e15492.
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
doi:10.1371/journal.pone.0015492
PMCID: PMC2975710  PMID: 21079746
22.  Preservation of Serratia marcescens by High-Vacuum Lyophilization 
Applied Microbiology  1966;14(4):561-567.
Water-washed Serratia marcescens (ATCC strain 14041) cells were lyophilized in an all-glass system capable of evacuation to pressures of less than 5 × 10-6 torr. Lyophilization at the lowest pressures resulted in 50 to 65% survival for unstabilized washed organisms compared with 10 to 20% when the cells were lyophilized at pressures of about 2.5 × 10-2 torr. At the latter pressures, 45 to 65% survival was obtained when NaCl or Naylor-Smith stabilizer was added to the cell suspensions before lyophilization. However, the stabilizers failed to increase significantly the levels of survival compared with water suspension when cells were lyophilized at pressures less than 10-5 torr. The high survival rates obtained by the high-vacuum technique may be attributed to the reduction of traces of molecular oxygen which has been reported to be destructive of the dried bacteria. Survival of unstabilized dried S. marcescens after 1-day storage increased markedly with decreasing sealing pressure. Under the highest vacuum attained, survival of the dried bacteria was not impaired by storage for up to 1 month at Dry Ice temperatures; at higher temperatures, viability losses occurred. Exposure of the dried unstabilized bacteria to dry air resulted in rapid viability loss. The inactivation could be stopped almost immediately by evacuation to pressures of less than 10-5 torr, but the evacuation failed to reverse the viability losses that occurred during exposure.
PMCID: PMC546782  PMID: 5332950
23.  Effect of Cholesterol on the Properties of Spray-Dried Lysozyme-Loaded Liposomal Powders 
AAPS PharmSciTech  2010;11(2):832-842.
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.
doi:10.1208/s12249-010-9442-8
PMCID: PMC2902320  PMID: 20464536
cholesterol; liposomes; lysozyme; spray-dried powders
24.  Phase I Trial of Intravesical Recombinant Adenovirus-Mediated Interferon-α2b Formulated in Syn3 for BCG failures in Non-Muscle-Invasive Bladder Cancer 
The Journal of urology  2013;190(3):850-856.
Purpose
A Phase l trial of intravesical recombinant adenovirus-mediated interferon-α2b gene therapy (rAd-IFNα) formulated with the excipient SCH Syn3 was conducted in patients with non-muscle invasive bladder cancer (NMIBC) who recurred after Bacillus Calmette-Guerin (BCG). The primary objective was to determine the safety of rAd-IFNα/Syn3; secondary endpoints were to demonstrate effective rAd-IFNα gene expression and preliminary evidence of clinical activity at three months.
Patients and Methods
Seventeen patients with recurrent NMIBC after BCG were enrolled. A single treatment of rAd-IFNα (3×109 to 3×1011 particles/mL) formulated with the excipient Syn3 was administered. Patient safety was evaluated for ≥12 weeks. Efficacy of gene transfer was determined by urine IFNα protein concentrations. Preliminary drug efficacy was determined at 3 months.
Results
Intravesical rAd-IFNα/Syn3 was well tolerated as no dose limiting toxicity (DLT) was encountered. Urgency was the most common adverse event and all were grade 1 or 2. rAd-IFNα DNA was not detected in the blood, however, transient low serum IFNα and Syn3 levels were measured. High and prolonged dose-related urine IFNα levels were achieved with the initial treatment. Of the 14 patients treated at doses ≥ 1010 particles/mL with detectable urine IFNα, 6 (43%) experienced a complete response at 3 months and 2 remained disease free at 29.0 and 39.2 months respectively.
Conclusion
Intravesical rAd-IFNα/Syn3 was well tolerated with no DLT encountered. Dose dependent urinary IFNα concentrations confirmed efficient gene transfer and expression. Intravesical rAd-IFNα/Syn3 demonstrated promising clinical activity in NMIBC recurring after BCG.
doi:10.1016/j.juro.2013.03.030
PMCID: PMC3951790  PMID: 23507396
gene therapy; bladder cancer; interferon
25.  Optimising biocatalyst design for obtaining high transesterification activity by α-chymotrypsin in non-aqueous media 
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.
doi:10.1186/1752-153X-2-2
PMCID: PMC2262082  PMID: 18269743

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