Comparative assessment of in vitro release kinetics of calcitonin polypeptide from biodegradable microspheres

The objective of our study was to compare the in vitro release kinetics of a sustained-release injectable microsphere formulation of the polypeptide drug, calcitonin (CT), to optimize the characteristics of drug release from poly-(lactide-co-glycolide) (PLGA) copolymer biodegradable microspheres. A modified solvent evaporation and double emulsion technique was used to prepare the microspheres. Release kinetic studies were carried out in silanized tubes and dialysis bags, whereby microspheres were suspended and incubated in phosphate buffered saline, sampled at fixed intervals, and analyzed for drug content using a modified Lowry protein assay procedure. An initial burst was observed whereby about 50% of the total dose of the drug was released from the microspheres within 24 hr and 75% within 3 days. This was followed by a period of slow release over a period of 3 weeks in which another 10-15% of drug was released. Drug release from the dialysis bags was more gradual, and 50% CT was released only after 4 days and 75% after 12 days of release. Scanning electron micrographs revealed spherical particles with channel-like structures and a porous surface after being suspended in an aqueous solution for 5 days. Differential scanning calorimetric studies revealed that CT was present as a mix of amorphous and crystalline forms within the microspheres. Overall, these studies demonstrated that sustained release of CT from PLGA microspheres over a 3-week period is feasible and that release of drug from dialysis bags was more predictable than from tubes.     

Applicability of various amphiphilic polymers to the modification of protein release kinetics from biodegradable reservoir-type microspheres.

The effects of various amphiphilic polymers on the kinetics of protein release from reservoir-type microspheres, prepared by a solid-in-oil-in-water emulsion-solvent evaporation method, were investigated. Bovine serum albumin (BSA), as a model protein, was firstly micronized through co-lyophilization with amphiphilic polymers, such as poly (ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), and pluronic F68. This process was based on the aqueous phase separation of protein and amphiphilic polymer induced by freezing-condensation. Mixing of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) (at a ratio of 4:6) in a methylene chloride solution provided a'polymer-alloy' structure, where the preformed solid BSA microparticles were selectively distributed in the inner PLGA-rich phase. The reservoir-type microspheres obtained through this process showed high entrapment efficiencies (more than 85%) and reduced initial burst releases (less than 10%). Although PVP did not modify the BSA release profile, PEG and pluronic F68 enhanced the BSA release, with no increase of the initial burst effect, responding to their loading percentage: 3% loading of PEG or pluronic F68 resulted in typical zero-order release kinetics. The abilities of these amphiphilic polymers to modify the protein release profile could be predicted from their partitioning characteristics in the polymer-alloys and in the methylene chloride/water system.     

Radiolabelled biodegradable microspheres for lung imaging.

The effect on lung accumulation of modifying the surface compositions of (99m)Tc poly(lactide-co-glycolide) (PLGA) and (99m)Tc poly(ethylene glycol)-poly(lactide-co-glycolide) (PEG-PLGA) microspheres with different surfactants was assessed after intravenous injection into rats. Microspheres were prepared with PLGA or PEG-PLGA by the emulsion solvent evaporation method using polyvinyl alcohol (PVA), polyethylene glycol (PEG), albumin (BSA) or poloxamer 188 as surfactant, in the external aqueous phase. Commercial human albumin microspheres (Sferotec((R)), HAM) were used as reference. According to the European Pharmacopeia, >80% of (99m)Tc-HAM in the size range 10-50 microm, must be accumulated in the lung 15 min after intravenous administration. By modifying the surfactant, the resulting lung accumulation was 99% for (99m)Tc-HAM, and more than 50% for PLGA microspheres prepared with poloxamer 188 (1 and 4%), reaching 67% with 8% Poloxamer 188 and around 30-39% for PLGA and PEG-PLGA microspheres prepared with the other surfactants. PLGA microspheres made with 8% poloxamer 188 gave good quality lung images under a gamma camera for the first few minutes, subsequently liver radioactivity masked lung images.     

Solvent influence on spray-dried biodegradable microspheres.

Microspheres of fixed poly(D,L-lactic acid) (PDLLA) composition--Resomer R104:R202H (30:70)--containing 20% w/w rifampicin have been spray-dried from a range of acetonic, halogenated, and solvent mixtures thereof under constant process conditions to examine the influence of solvent selection on microsphere characteristics. Solubility of the polymer composite in the studied solvents determined the kinetics of polymer deposition during drying. Viscosity studies provided an indirect index of solvent power in ascending order: acetone (ACT) < dichloromethane (DCM) < chloroform (CFM) < halothane (HAL). Accordingly, poorer acetonic solvents produced a more open, porous matrix of increased mean diameter, whereas DCM, CFM and HAL generated more coherent matrices of greater density and elevated glass transition temperature, which significantly retarded drug release. Yield generally increased in parallel with solvent strength and microsphere density consistent with the proposed generalized particle formation mechanism. Residual solvent also increased with particle density, both parameters being interrelated and dictated by the inherent affinity of the polymer composite for individual solvents. In turn, the position of glass transition temperature (Tg) and the quantity of associated polymer stress-relaxation were a direct function of amount and persistence of organic residue. The magnitude of these changes determined the relative rates and extents of microsphere ageing, as measured by drug release studies. In general, rate of drug release increased with Tg, after corrections were made for specific surface area (r2 = 0.963). Overall, solvent choice for spray-drying has a remarkable influence on microsphere characteristics and, accordingly, technological as well as toxicological considerations should be paid during selection of same.     

Methods for study of fluphenazine kinetics in man.

Fluphenazine and its principal metabolites, fluphenazine sulphoxide, and 7-hydroxyfluphenazine were identified and quantified in human plasma, urine and faeces following intramuscular and oral administration of 14C-fluphenazine dihydrochloride. The presence of a conjugate fraction was also noted. Unmetabolized fluphenazine was selectively extracted into n-heptane. The metabolites were separated by solvent extraction into toluene. Conjugates were hydrolysed back to fluphenazine, fluphenazne sulphoxide and 7-hydroxyfluphenazine. Fluphenazine and fluphenazine conjugates were also measured in the urine of patients receiving long term non-radioactive fluphenazine decanoate therapy. The urinary excretion rate of the conjugate fraction was systematically related to the plasma concentration, regardless of urine flow rate or pH, providing a convenient method for the assessment of fluphenazine kinetics by urinary excretion studies not involving administration of labelled drug.     

Release kinetics and immunogenicity of parvovirus microencapsulated in PLA/PLGA microspheres

The aim of this work was to examine the immunogenicity of microencapsulated inactivated duck parvovirus in Muscovy duck (Cairina moschata) and goose. Inactivated duck parvovirus suspension was microencapsulated into 14-17 kDa poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA50:50H) by coacervation. The in vitro antigen release from individual and mixed PLA and PLGA50:50H microspheres (MS) was biphasic with an initial lag-phase of approx. 10 days followed by a relatively constant release over additional 12 days. By varying the composition of PLA+PLGA50:50H MS mixtures from 3+1 to 1+3, the release kinetics could be altered and controlled efficiently. The antigen-loaded MS were injected subcutaneously into ducks. The immune response, expressed as virus neutralisation (VN) titres, after single administration of MS was modest, i.e. below 200 over the 6 weeks tested, unless the animals were pre-immunised 3 weeks before injecting the MS. The weak immune response was attributed to the low dose injected and inappropriate antigen release kinetics. With pre-immunised animals, however, the results were encouraging and showed that the encapsulated parvovirus was immunogenic.     

Removal of chloroform from biodegradable therapeutic microspheres by radiolysis

Radioactive holmium-166 loaded poly(l-lactic acid) microspheres are promising systems for the treatment of liver malignancies. These microspheres are loaded with holmium acetylacetonate (HoAcAc) and prepared by a solvent evaporation method using chloroform. After preparation the microspheres (Ho-PLLA-MS) are activated by neutron irradiation in a nuclear reactor. It was observed that relatively large amounts of residual chloroform (1000-6000 ppm) remained in the microspheres before neutron irradiation. Since it is known that chloroform is susceptible for high-energy radiation, we investigated whether neutron and gamma irradiation could result in the removal of residual chloroform in HoAcAc-loaded and placebo PLLA-MS by radiolysis. To investigate this, microspheres with relatively high and low amounts of residual chloroform were subjected to irradiation. The effect of irradiation on the residual chloroform levels as well as other microsphere characteristics (morphology, size, crystallinity, molecular weight of PLLA and degradation products) were evaluated. No chloroform in the microspheres could be detected after neutron irradiation. This was also seen for gamma irradiation at a dose of 200 kGy phosgene, which can be formed as the result of radiolysis of chloroform, was not detected with gas chromatography-mass spectrometry (GC-MS). A precipitation titration showed that radiolysis of chloroform resulted in the formation of chloride. Gel permeation chromatography and differential scanning calorimetry showed a decrease in molecular weight of PLLA and crystallinity, respectively. However, no differences were observed between irradiated microsphere samples with high and low initial amounts of chloroform. In conclusion, this study demonstrates that neutron and gamma irradiation results in the removal of residual chloroform in PLLA-microspheres.     

An analysis of first-order release kinetics from albumin microspheres.

Release rates from BSA microspheres prepared from various conditions are analysed using a previously reported equation expressing the first-order release rate constant. The permeability constants calculated applying the equation on experimental release rates are in good agreement with the constants measured from permeation studies using planar membrane, for various preparation conditions. It is shown that the equation expressing the first-order release rate constant is valid more extensively. The permeability constant varies depending on the preparation conditions, and the reason for variation is shown clearly to be the difference in degree of swelling of the polymer. It was found from regression analysis that there is relatively simple correlation between unknown parameters of the equation and the preparation conditions. Release rate constants can be calculated applying the equation on the known parameters and the estimated values of the unknown parameters from the correlation. Good agreement was found between the calculated values and experimental ones; therefore, at least as far as we examined here, the release rate constant of the microsphere can be estimated from the preparation conditions.     

Controlled release of anti-cocaine catalytic antibody from biodegradable polymer microspheres

Recent reports have shown that anti-cocaine catalytic monoclonal antibody 15A10 reduces the toxic effect of cocaine by increasing its breakdown to systemically inert products ecgonine methylester and benzoic acid. This study reports the microencapsulation of antibody 15A10 using biodegradable poly (lactic-glycolic) acid (PLGA) by double emulsion technique. Formulation parameters such as protein loading, polymer molecular weight and the presence of zinc carbonate were studied for their effects on in-vitro release of antibody from microspheres. The initial burst release was decreased by the reduction of the protein (as % of total ingredients) in the formulation. Although changing the polymer molecular weight did not cause a reduction in initial burst release, it was effective in improving the release rate. The inclusion of zinc carbonate in microsphere preparation resulted in increase in initial burst release. An in-vivo study in mice revealed the presence of antibody in blood up to ten days following subcutaneous injections. These data demonstrate a potential for a sustained-release formulation of monoclonal antibody 15A10 for treatment of cocaine addiction.     

Protein loaded biodegradable microspheres based on PLGA-protein bioconjugates.

Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was chemically conjugated to lysozyme, a model protein drug, by coupling a terminal carboxylic acid in PLGA with primary amine groups present in lysozyme. The conjugation was carried out in dimethylsulphoxide (DMSO) by using carbodiimide as a coupling agent. The PLGA-lysozyme conjugate, dissolved in a co-solvent system of DMSO and methylene chloride, was directly formulated into microspheres by an oil-in-water (O/W) single emulsion solvent evaporation technique. Morphological characteristics of the resultant microspheres, loading efficiencies, and protein release behaviours with protein instability problems were investigated in comparison with those of the microspheres prepared by water-in-oil-water (W/O/W) double emulsion and O/W single emulsion techniques which employed PLGA with unconjugated lysozyme for the formulation.     

Immunogenicity of meningococcal PorA formulations encapsulated in biodegradable microspheres.

The purpose of our study was to investigate the possibility to microencapsulate liposomes and meningococcal outer membrane vesicles (OMV), both containing neisserial pore protein A (PorA), in biodegradable dextran- and mannan-based microspheres and to study the immunogenicity of the encapsulated PorA formulations. PorA-liposomes and OMV were encapsulated in dextran- or mannan-based microspheres by using an aqueous two-phase system consisting of a polyethylene glycol solution and a methacrylated dextran or mannan solution. The formulations were characterized for size distribution, PorA structure and antigen recovery after release. Calcein-containing model liposomes were used to establish the encapsulation efficiency and release profiles from both types of microspheres. The immunogenicity of the PorA-containing formulations was determined in mice after subcutaneous immunization. Liposomes were encapsulated in dextran and mannan microspheres with a high efficiency (70-90%). Calcein liposomes, after a 5-day lag period, exhibited apparent zero-order release kinetics from both types of microspheres between Days 5 and 10 of incubation in vitro. The total release was 80 and 100% from mannan and dextran microspheres, respectively. The trimeric PorA conformation was preserved in the released liposomes and OMV and the antigen was partly recovered. The immunogenicity of PorA-liposomes and OMV encapsulated in dextran or mannan microspheres was preserved. In conclusion, PorA-liposomes and OMV could be encapsulated in dextran- and mannan-based microspheres with high efficiency. The immunogenicity of encapsulated antigen was preserved.     

Effect of morphological properties on drug release from biodegradable microspheres]

The morphological properties of poly(beta-hydroxybutyric acid) (PHB) or poly(L-lactic acid) microspheres loading flomoxef sodium (FMOX) were investigated with regard to FMOX release. The release profiles of FMOX from the microspheres could be divided into two types, a sustained release type and a burst one. Two representative PHB microspheres, the release profiles of which were quite different from those of FMOX, were compared in detail from a morphological point of view. The shapes of their surfaces and sections were observed by using scanning electron microscopy (SEM), and FMOX distribution was analyzed by using electron probe microanalysis. The crystallinity of polymers was further measured by powder X-ray diffratometry. There was little difference in the FMOX distribution and their microscopic properties such as sphere size, specific surface area, shape of surface and section. In contrast, water penetration into the inside of the microspheres was found to be clearly different by use of cryogenic SEM. A significant difference was also observed in the crystallinity of polymers forming the microspheres. The release of FMOX from the microspheres was affected by the crystallinity of polymers forming the microspheres, and burst phenomena occurred in case the polymer was highly crystallized. It was speculated that the crystallization of polymer induced micro voids in the microspheres which functioned as channels for water penetration.     

Release kinetics of catechins from chewing gum

Chewing gums containing catechins were prepared by applying a novel solid dispersion and hot-melt fluid bed coating method. The effect of varying levels of Eudragit coating to the granules on the prolonged release of catechins from chewing gum was investigated. In vivo release profiles of catechins from the formulations were investigated to determine its release kinetics. Dissolution data were used as the primary basis for comparison. The equations with zero order, first-order, or square root of time release kinetics were used to fit data. The results showed that the releases of catechins from chewing gum were well extended with the growing coating level of Eudragit to the granules. The statistical analysis using nonlinear regression software suggested that the Higuchi equation describes the data better than others. The relatively high degree of fit using the Higuchi equation implies that the kinetic process is involved in the release of drug from the dosage form. A possible release mechanism based on this profile is presented in this report. This approach suggested the potential of using chewing gum as a promising controlled-release delivery system of drugs. The effect of polyvinyl acetates with different crosslinkings on the release profiles of catechins was also studied.     

Solvent influence on spray-dried biodegradable microspheres

Microspheres of fixed poly(D,L-lactic acid) (PDLLA) composition Resomer R104:R202H (30:70) containing 20% w/w rifampicin have been spray-dried from a range of acetonic, halogenated, and solvent mixtures thereof under constant process conditions to examine the influence of solvent selection on microsphere characteristics. Solubility of the polymer composite in the studied solvents determined the kinetics of polymer deposition during drying. Viscosity studies provided an indirect index of solvent power in ascending order: acetone (ACT) &lt; dichloromethane (DCM) &lt; chloroform (CFM) &lt; halothane (HAL). Accordingly, poorer acetonic solvents produced a more open, porous matrix of increased mean diameter, whereas DCM, CFM and HAL generated more coherent matrices of greater density and elevated glass transition temperature, which significantly retarded drug release. Yield generally increased in parallel with solvent strength and microsphere density consistent with the proposed generalized particle formation mechanism. Residual solvent also increased with particle density, both parameters being interrelated and dictated by the inherent affinity of the polymer composite for individual solvents. In turn, the position of glass transition temperature (Tg) and the quantity of associated polymer stressrelaxation were a direct function of amount and persistence of organic residue. The magnitude of these changes determined the relative rates and extents of microsphere ageing, as measured by drug release studies. In general, rate of drug release increased with Tg, after corrections were made for specific surface area (r2=0.963). Overall, solvent choice for spray-drying has a remarkable influence on microsphere characteristics and, accordingly, technological as well as toxicological considerations should be paid during selection of same.     

Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres.

Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson's disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30mum, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive.     

Cyclophosphamide loaded albumin microspheres II. Release characteristics

The purpose of this investigation was to evaluate the release characteristics of cyclophosphamide (CP) from glutaraldehyde stabilized human serum albumin microspheres, and to study the effect of the extent of cross-linking, the amount of the stabilizing agent and the size of the microspheres on the in vitro release of CP. Microspheres were prepared by emulsion polymerization method using two different volumes (0.1 and 0.7 ml) of glutaraldehyde solution (25 per cent) and two different crosslinking durations (15 min and 1 h). The resulting mean particle size of the microspheres also varied between 2.5 microns and 3.7 microns. The total CP content in microspheres was analysed from the surface drug and the entrapped drug.     

In vitro and in vivo characterization of biodegradable enoxacin microspheres.

The in vitro release and plasma concentration profiles of sustained release enoxacin microspheres intended for the treatment of bone and systemic infections due to sensitive strains of bacteria were investigated. Microspheres of enoxacin were prepared by using poly(glycolic acid-co-DL-lactic acid) (PLGA) by the emulsion solvent evaporation technique and characterized by in vitro release in an incubator, and in vivo release in the rat subcutaneous model. The microspheres were spherical in nature, and particle size range had a significant influence on the in vitro release. The enoxacin plasma concentration 2 h after the administration of treatments was two-fold higher in animals who received the free drug compared with those who received microspheres of size range 125-250 microm. The plasma of animals who received the free drug was depleted of enoxacin by the end of the first day. However, the plasma concentration of enoxacin in the animals who received microspheres was sustained above 0.5 microg/ml for about 8 days. The results show that biodegradable microspheres of enoxacin can be prepared which release the antibiotic in vivo for days following a subcutaneous administration. This should provide a means for the sustained treatment of infections due to sensitive strains of bacteria.     

Release of small water-soluble drugs from multiblock copolymer microspheres: a feasibility study.

Poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) multiblock copolymer was investigated as a possible matrix for controlled delivery of small water-soluble drugs. Two molecules were selected as sustained release candidates from microspheres: leuprorelin acetate (peptide of Mw = 1270 D) and vitamin B(12) (Mw = 1355 D). First, vitamin B(12)-loaded microspheres were prepared using a double emulsion method and preparation parameters were varied (surfactant in the first emulsion and copolymer composition). The resulting microsphere structure, entrapment efficiency and release rate were evaluated. Vitamin B(12)-loaded microsphere parameters could easily be tailored to achieve specific requirements. The addition of surfactant in the first preparation process led to a significant increase of the microsphere entrapment efficiency, whereas the decrease of the PEGT copolymer content allowed the release rates from microspheres to be precisely decreased. However, leuprorelin acetate-loaded microspheres did not show the same characteristics when prepared with the same parameters, possibly because of a high water solubility discrepancy between the vitamin B(12) and the peptide. This study shows the suitability of PEGT/PBT microspheres as a controlled release system for vitamin B(12), but not for leuprorelin acetate. It also underlines the necessity of tailored development for each individual drug and emphasizes the risk of using model molecules.     

New drug salt formation in biodegradable microspheres

PURPOSE: To investigate the effects of inorganic salts in the external phase of an oil-in-water (O/W) emulsion method during microsphere preparation. METHODS: An O/W emulsion method was used to prepare poly(D,L-lactic acid) microspheres containing quinidine sulfate. Different inorganic salts were used in the external phase during microsphere preparation. Microsphere drug loading was determined by UV and the drug salt anions inside the microspheres were determined by ion chromatography. RESULTS: New drug salts were formed during encapsulation in the microspheres when salts with non-common anions to the drug salt were used. Drug loading increased when NaClO4 or NaSCN were used. The fraction of drug as the new salt in microspheres increased non-linearly with the salt concentration in the external phase, however, the fraction of drug as the new encapsulated salt was linearly related to drug loading. Drug loading decreased and new salt fraction increased with increasing organic solvent volume or with decreasing cosolvent polarity. CONCLUSIONS: Introducing salts containing non-common anions to the drug salt employed in the external phase of O/W emulsion microsphere method leads to new salt formation. The extent of new drug salt formation is affected by salt levels added, cosolvent type and polymer concentration.     

Release of mifepristone from biodegradable matrices: experimental and theoretical evaluations

Diffusion of mifepristone in poly [(D,L) lactide-co-glycolide)] films was studied by release experiments. Five 50/50 copolymers of increasing molecular weights were used. The degradation effects were shown by gel permeation chromatography (GPC). Release kinetics show the effect of copolymer molecular weights on diffusion and degradation properties of loaded films. A new theoretical model for drug release from a biodegradable matrix was proposed with two assumptions: correlation of the diffusion coefficient with the polymer molecular weight and existence of a first order degradation kinetic. Higuchi's equation is verified at early time and the diffusion coefficient in the non-degraded polymer can be measured. The degradation constant is determined at long time and is compared with the results of GPC.