Preparation and characterization of protein loaded microspheres based on a hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid).

The purpose of this study was to investigate the suitability of a novel hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid) (PLHMGA), as controlled release system for pharmaceutical proteins. Dextran Blue (as a macromolecular model compound) and lysozyme-loaded PLHMGA and PLGA (control formulation) microspheres were prepared by a solvent evaporation technique. The Dextran Blue and lysozyme loaded PLHMGA microspheres prepared with 10% polymer solution showed, because of a high porosity, a high burst release (35-75%) and the remaining content was released in a sustained manner for 15-20 days. The microspheres prepared with 15 and 20% polymer solution had a lower porosity and showed a pulsed release after day 8 and in 27 days they released more than 90% of Blue Dextran. The release of lysozyme was incomplete, likely due to aggregation of part of the encapsulated protein. Spectroscopic analysis of the released lysozyme indicated fully preserved secondary/tertiary structure and an enzyme activity assay showed that the specific activity of the released protein was maintained. An in vitro degradation study showed that the release of Blue Dextran and lysozyme is essentially controlled by the degradation of the microspheres. This study shows that microspheres made of the hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid), are promising systems for the controlled release of pharmaceutical proteins.

Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression.

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size 100 and 120nm, respectively) and almost neutral particles. In vitro tissue culture experiments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as compared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added to the transfection medium, only the transfection activity of the targeted polyplexes was reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor. Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes. While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations of 0.09microg/mL, the PEGylated systems could be incubated at ten times higher concentration before aggregation occurred indicating excellent shielding of the surface charge of the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes makes them interesting for in vivo applications.

Homogeneous nucleation of water in mesoporous zeolite cavities.

In this paper, we show that water inside mesoporous cavities in zeolites can be supercooled to ca. -40 degrees C at which point homogeneous nucleation of the water takes place. The fundamental phenomena observed here are similar to those reported earlier in for example emulsion droplets or droplets in the vapor phase. However, as these zeolite materials are widely available, they may provide an easily accessible source for studies of supercooled liquids in confinements. Next to this, it is now possible to discriminate with thermoporometry between mesoporous cavities inside the zeolite crystals, in which homogeneous nucleation takes place, and mesopores that are connected to the external surface in which heterogeneous nucleation takes place.

Pharmaceutical development of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum anticancer agent AP 5280.

AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water. However, as aqueous solutions of AP 5280 proved to be labile upon sterilization by moist heat, it was decided to develop a lyophilized dosage form. Initially, glass vials were used as primary packaging, but this led to a high breakage rate, which could be completely prevented by the use of CZ resin vials. Stability studies to date show that the lyophilized product in glass vials is stable for at least 12 months when stored at 2-8 degrees C in the dark and the lyophilized product in CZ resin vials is stable for at least 6 months under these conditions. Photostability testing revealed photolability of AP 5280 drug substance and lyophilized product in both types of primary container, necessitating storage in the dark. The first clinical experiences indicate that the proposed formulation is fully applicable for use in the clinical setting.

Pharmaceutical development of a parenteral lyophilized formulation of the antimetastatic ruthenium complex NAMI-A.

This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat. The effect of several excipients on the stability of the formulation solution was investigated. None of them provided sufficient stability to allow long-term storage of an aqueous solution of NAMI-A. Therefore, a lyophilized product was developed. Five different formulations were prepared and subjected to thermogravimetric (TG) analysis and stability studies at various conditions for 1 year. Minimal degradation during the production process is achieved with a formulation solution of pH 3-4. Of the acids tested, only hydrochloric acid (HCl 0.1 mM) both stabilized the formulation solution and was compatible with the lyophilized product. This product was stable for at least 1 year when stored at -20 degrees C, 25 degrees C/60% relative humidity (RH) and 40 degrees C/75% RH, and was also photostable.

Development of a lyophilized parenteral pharmaceutical formulation of the investigational polypeptide marine anticancer agent kahalalide F.

Kahalalide F is a novel antitumor agent isolated from the marine mollusk Elysia rufescens; it has shown highly selective in vitro activity against androgen-independent prostate tumors. The purpose of this study was to develop a stable parenteral formulation of kahalalide F to be used in early clinical trials. Solubility and stability of kahalalide F were studied as a function of polysorbate 80 (0.1%-0.5% w/v) and citric acid monohydrate (15-15 mM) concentrations using an experimental design approach. Stabilities of kahalalide F lyophilized products containing crystalline (mannitol) or amorphous (sucrose) bulking agents were studied at +5 degrees C and +30 degrees C +/- 60% relative humidity (RH) in the dark. Lyophilized products were characterized by infrared (IR) spectroscopy and differential scanning calorimetry (DSC). Recovery studies after reconstitution of kahalalide F lyophilized product and further dilution in infusion fluid were carried out to select an optimal reconstitution vehicle. It was found that a combination of polysorbate 80 and citric acid monohydrate is necessary to solubilize kahalalide F. Lyophilized products were considerably less stable with increasing polysorbate 80 and citric acid monohydrate concentrations, with polysorbate 80 being the major effector. A combination of 0.1% w/v polysorbate 80 and 5 mM citric acid monohydrate was selected for further investigation. Lyophilized products containing sucrose as a hulking agent were more stable compared to the products containing mannitol. The glass transition temperature of the sucrose-based product was determined to be + 46 degrees C. The amorphous state of the product was confirmed by IR analysis. A solution composed of Cremophor EL, ethanol, and water for injection (5%/5%/90% v/v/v CEW, kept kahalalide F in solution after reconstitution andfurther dilution with 0.9% w/v sodium chloride (normal saline) to 1.5 microg/m. A stable lyophilized formulation was presented containing 100 microg of kahalalide F, 100 mg sucrose, 2.1 mg citric acid monohydrate, and 2mg polysorbate 80 to be reconstituted with a vehicle composed of 5%/5%/90% v/v/v CEW and to be diluted further using normal saline.

Characterization of poly(L-lactic acid) microspheres loaded with holmium acetylacetonate.

Holmium-loaded PLLA microspheres are useful systems in radioembolization therapy of liver metastases because of their low density, biodegradability and favourable radiation characteristics. Neutron activated Ho-loaded microspheres showed a surprisingly low release of the relatively small holmium complex. In this paper factors responsible for this behaviour are investigated, in particular by the use of differential scanning calorimetry, scanning electron microscopy, infrared spectroscopy and X-ray diffraction. The holmium complex is soluble in PLLA up to 8% in films and 17% in microspheres. Interactions between carbonyl groups of PLLA, and the Ho-ion in the HoAcAc complex, explain very satisfactorily the high stability of holmium-loaded microspheres.

Formation of dextran hydrogels by crystallization.

In this paper, a novel method is presented for the preparation of dextran hydrogels and microspheres, based on crystallization. Although dextrans are known to be well soluble in water, precipitation was observed in concentrated aqueous solutions of low molecular weight dextran (dextran 6000), whereas for solutions of dextran with higher molecular weights (dextran 40,000 and 220,000) no precipitation was observed in the time-frame studied. The kinetics of the precipitation process were studied and showed that precipitation was faster when more concentrated dextran solutions were used. Furthermore, the precipitation process was accelerated by stirring and by the presence of salts. Depending on the precipitation time, microspheres or gels were obtained. The precipitates were insoluble in water at room temperature, but readily dissolved in boiling water or DMSO. IR spectroscopy and (modulated) differential scanning calorimetry ((M)DSC) demonstrated that the precipitates were crystalline. We hypothesize that crystallization is due to association of the chains through hydrogen bonding, induced by the large polymer/water ratio in concentrated dextran 6000 solutions.

Pharmaceutical development of a parenteral lyophilized formulation of the novel antitumor agent aplidine.

Aplidine is a naturally occurring cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. Aplidine displays promising in vitro and in vivo antitumor activities against various solid human tumor xenografts and is therefore developed now for clinical testing. The aim of this study was to develop a stable parenteral pharmaceutical dosage form for clinical Phase I testing. Aplidine raw material was characterized by using several chromatographic and spectrometric techniques. These experiments showed that aplidine exists as two isomers. A stability-indicating HPLC assay was developed. Solubility testing showed that aplidine exhibits very poor aqueous solubility. Because solubilized aplidine showed substantial degradation under heat and light stress testing conditions, it was decided to develop a lyophilized dosage form. Freeze-drying was carried out with a 500 micrograms/mL solution of aplidine in 40% (v/v) tert-butanol in Water for Injection (WfI) containing 25 mg/mL D-mannitol as a bulking agent. Differential scanning calorimetry was applied to determine the optimal freeze-drying cycle parameters. The prototype, containing 500 micrograms aplidine and 25 mg D-mannitol per vial, was found to be the optimal formulation in terms of solubility, length of lyophilization cycle, and dosage requirements in the forthcoming Phase I clinical studies. Quality control of the freeze-dried formulation demonstrates that the manufacturing process does not affect the integrity of aplidine. The optimal reconstitution solution was found to be 15/15/70% (v/v/v) Cremophor EL/ethanol/WfI (CEW). Both reconstituted product and dilutions of the reconstituted product with normal saline (up to 1:100 v/v) appeared to be stable for at least 24 hours after preparation. Shelf-life data, available thus far, show that the lyophilized formulation is stable for at least 1 year when stored at +2-8 degrees C in the dark.

Long term stability of poly((2-dimethylamino)ethyl methacrylate)-based gene delivery systems.

PURPOSE: To study the stability of polymer-plasmid complexes (polyplexes) both as an aqueous dispersion and in their lyophilized form. METHODS: The characteristics of the polyplexes (size, charge and transfection potential) were monitored at different temperatures. Moreover, we studied possible changes in the secondary and tertiary structure of the plasmid by agarose gel electrophoresis and by CD spectroscopy to gain insight into the mechanism of polyplex degradation. RESULTS: The polyplexes preserved almost their full transfection potential after aging in an aqueous solution of 20 mM Hepes (pH 7.4) containing 10% sucrose at 4 and 20 degrees C for 10 months. On the other hand, the polyplexes aged at 40 degrees C were rather unstable and lost their transfection capability with a half-life of around 2 months. During storage, conformational changes in the secondary and tertiary structure of DNA were observed. When naked plasmid DNA was aged at 40 degrees C as an aqueous solution and complexed with polymer just before the transfection experiment, a slower drop in its transfection capability was observed. The freeze-dried polyplexes using sucrose as lyoprotectant almost fully retained their transfection efficiency, even when aged at 40 degrees C for 10 months. CONCLUSIONS: This study provides information about polyplex stability in aqueous dispersions on storage and demonstrates that freeze-drying is an excellent method to ensure long term stability.

Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes.

In this paper the effect of the topology of plasmid DNA (supercoiled, open-circular and linear) on its binding characteristics with the polymeric transfectant poly((2-dimethylamino)ethyl methacrylate) was studied. The formed polyplexes were also evaluated for their transfection properties in vitro in two different cell lines. Anion-exchange chromatography was used for the separation of supercoiled and open-circular plasmid from a plasmid stock solution. Linear plasmids were prepared by endonucleases that cleaved the plasmid either in the promoter region or in a region not specific for expression (ampicillin resistance region). Plasmid DNA was also heat-denatured for 6 h at 70 degrees C, resulting in DNA mainly in the open-circular and oligomeric forms. The transfection of two different cell lines was dependent on the topology of the DNA in the order supercoiled>open-circular approximately heat-denatured>linear DNA prepared by cleaving in the nonspecific region>linear DNA prepared by cleaving in the promoter region. No differences in the size of the complexes or in the quenching of the DNA-intercalating fluorophore acridine orange were found as function of the topology. However, circular dichroism spectroscopy revealed differences between the topological plasmid species, both in the free form and in the presence of excess of cationic polymer.

The effect of formulation parameters on the size of poly-((2-dimethylamino)ethyl methacrylate)-plasmid complexes.

The aim of this study was to gain insight into the formulation parameters affecting the size of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes (polyplexes). Experimental designs were applied to screen and optimize several variables, which may influence the complex size. In a screening design, it was demonstrated that at a fixed concentration of plasmid (40 micrograms/ml) after incubation with polymer, the size of the resulting polyplexes was highly dependent on the polymer/plasmid ratio as well as on the pH, viscosity (i.e. sucrose concentration) and ionic strength of the aqueous solution. However, the temperature, PEG 600 (up to 5% (v/v)) and Tween 80 (up to 0.2%) had a marginal effect on the size of the polyplexes. In an optimization design, the effect of the pH, polymer/plasmid ratio and Tween on the size of the polymer/plasmid complexes prepared at relatively high concentration of plasmid (50-200 micrograms/ml) was evaluated. Based on the results of the optimization design, a mathematical model was derived, which describes the relationship between the size of the polyplexes and the different formulation parameters. This model shows that even at high plasmid concentration (200 micrograms/ml), small sized polyplexes were formed at low pH and ionic strength, especially when the solution contains 20% (w/v) sucrose. This concentrated polyplex dispersion (polymer/plasmid ratio > 3/1 (w/w), 200 micrograms plasmid/ml) can be diluted down to 5 micrograms/ml plasmid without significant changes in particle size and transfection potential. At lower ratios, a growth in particle size was observed upon dilution of the complexes, which might also explain the low transfection efficiency of these polyplexes in vitro.

Stabilization of polymer-based gene delivery systems.

To preserve the size and transfection potential of polymer-plasmid complexes. Freeze-drying and freeze-thawing were used for stabilization of these complexes. The concentration of the sugars is an important factor affecting both the size and transfection capability of the complexes after freeze-drying and freeze-thawing. However, the type of lyoprotectant (sugar) used is of minor importance. It is also shown that when damage to polymer-plasmid complexes occurs, it results from the drying process but is not due to the freezing step.

Pharmaceutical development of a parenteral lyophilized formulation of the investigational antitumor neuropeptide antagonist [Arg6, D-Trp7,9, MePhe8]-Substance P [6-11].

The aim of this study was to develop a stable parenteral dosage form for the investigational cytotoxic drug [Arg6, D-Trp79,MePhe8]-Substance P [6-11] (Substance P Antagonist G; Antagonist G). Antagonist G bulk drug was structurally and analytically characterized. The drug exhibits excellent aqueous solubility, although relatively poor aqueous stability characteristics. Lyophilization was, therefore, selected as the manufacturing process. Differential scanning calorimetry studies were conducted to determine the freeze-drying cycle parameters which resulted in a stable, lyophilized formulation of Antagonist G. The prototype, containing 50 mg Antagonist G per vial, was found to be the optimal formulation in terms of solubility, length of the freeze-drying cycle, stability, and dosage requirements in the planned phase I clinical trials. Quality control of the freeze-dried formulation showed that the manufacturing process does not change the integrity of Antagonist G. Shelf life studies demonstrated that the formulation is stable for at least 3 years, when stored at 2-8 degrees C in a dark environment. Oxidative degradation products of Antagonist G were isolated and structurally characterized by mass spectrometry, nuclear magnetic resonance spectroscopy, and infrared spectroscopy.

2-(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents.

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a water-soluble cationic polymer, which is able to bind to DNA by electrostatic interactions. At a polymer/plasmid ratio above 2 (w/w) positively charged complexes were formed with a size around 0.2 microm. The transfection efficiency of polymer/plasmid complexes was evaluated in cell culture (COS-7 and OVCAR-3 cells) using a pCMV-lacZ plasmid, encoding for beta-galactosidase, as a reporter gene. The optimal transfection efficiency was found at a PDMAEMA/plasmid ratio of 3-5 (w/w). Under these conditions 3-6% of the cells were actually transfected. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. A pronounced effect of the molecular weight of the polymer on the transfection efficiency was observed. An increasing molecular weight resulted in an increasing number of transfected cells. Dynamic light scattering experiments showed that high molecular weight polymers (Mw>300 kDa) were able to condense DNA effectively (particle size 0.15-0.20 microm). In contrast, when plasmid was incubated with low molecular weight PDMAEMA, large complexes were formed (size 0.5-1.0 microm). Copolymers of DMAEMA with methyl methacrylate (MMA), ethoxytriethylene glycol methacrylate (triEGMA) or N-vinyl-pyrrolidone (NVP) also acted as transfection agents. A copolymer with 20 mol % of MMA showed a reduced transfection efficiency and a substantial increased cytotoxicity compared with a homopolymer of the same molecular weight. A copolymer with triEGMA (48 mol %) showed both a reduced transfection efficiency and a reduced cytotoxicity, whereas a copolymer with NVP (54 mol %) showed an increased transfection efficiency and a decreased cytotoxicity as compared to a DMAEMA homopolymer.

Thermal analysis of freeze-dried liposome-carbohydrate mixtures with modulated temperature differential scanning calorimetry.

In this study we investigated the use of modulated temperature differential scanning calorimetry (MTDSC) for the detection of the glass transition temperature (Tg) in freeze-dried cakes of lyoprotected liposomes and for the analysis of frozen carbohydrate solutions. The glass transition appeared in the reversing heat flow, whereas the bilayer melting endotherm was observed in the nonreversing heat flow. This enabled the detection of Tg even in samples were the glass and bilayer transition overlapped. In addition, relaxation processes occurring in nonannealed freeze-dried carbohydrate-liposome mixtures, which hinder the determination of Tg with conventional DSC, were also separated from the heat capacity related heat flow. Analysis of frozen carbohydrate solutions with MTDSC facilitated the identification of the glass transition, devitrification peak, and "softening" transition, which could help to further optimize freeze-drying conditions by rationale. Sampling and selection of experimental parameters are discussed for the special case of porous, freeze-dried cakes.

Stabilization of gene delivery systems by freeze-drying.

Freeze-drying of three different forms of gene delivery systems was performed using a controlled two-step drying process and 10% sucrose as lyoprotectant. Complexes of pCMVL plasmid with transferrin-conjugated polyethylenimine, adenovirus-enhanced transferrinfection consisting of pCMVL/transferrin-polylysine complexes linked to inactivated adenovirus particles, and a recombinant, E1-defective adenovirus expressing a luciferase reporter gene were tested. Three weeks after freeze-drying the reagents were rehydrated with water and tested for transfection activity. Luciferase gene expression levels were retained at high levels in all three systems, in contrast to reagents stored in solution. The use of the lyoprotectant was essential. In the absence of sucrose the transfection activities dropped by a factor of 100-1000. The data suggest freeze-drying as a useful method for stabilization and storage of standardized batches of transfection agents.

Interactions between organic anions, micelles and vesicles in model bile systems.

Biliary lipid secretion probably involves both 'micellization' and 'vesiculization' of bile-canalicular membrane lipids. Several hydrophilic organic anions inhibit the secretion of lipids into the bile without altering bile salt secretion [Verkade, Vonk and Kuipers (1995) Hepatology 21, 1174-1189]. Hydrophobic organic anions do not interfere with biliary lipid secretion. We investigated whether the organic-anion-induced inhibition of biliary lipid secretion in vivo could be attributed to inhibition of micellization, by the application of in vitro models of micellization. Carboxyfluorescein was entrapped in a self-quenching concentration in small unilamellar vesicles (SUV) composed of cholesterol/egg phosphatidylcholine (molar ratios 0, 0.2 and 0.5). Certain organic anions clearly affected the bile-salt-induced release of fluorescence from these SUV, reflecting interference with micellization. However, the effects of hydrophilic and hydrophobic organic anions did not correspond with their effects on biliary lipid secretion in vivo, irrespective of the bile salt species used (taurocholate, taurodeoxycholate or tauroursodeoxycholate) and of the lipid composition of the SUV. Ultracentrifugation and dynamic light-scattering studies indicated that organic anions do interact with bile salt/ phosphatidylcholine/cholesterol mixed micelles, but that they do not inhibit micellization, for example by competing with phosphatidylcholine and/or cholesterol for incorporation into mixed micelles. In conclusion, the present in vitro data indicate that the in vivo mechanism of organic-anion-induced inhibition of biliary lipid secretion is not mediated by inhibition of micellization.