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Biomedical subjects

J Demeester

Publications and source records attributed to J Demeester.

At least 19 recordsLinked to original sources
PubMed2001

Structural alterations of gene complexes by cystic fibrosis sputum.

Gene complexes with optimal physicochemical characteristics for cystic fibrosis (CF) gene therapy in vitro may become inactive in vivo as a result of destruction upon interaction with CF mucus. Therefore, we examined in this study to what extent main sputum components (linear DNA, mucin, phosphatidylcholine, phosphatidylglycerol, and albumin) may disintegrate lipoplexes. We found that mixing linear DNA with lipoplexes, in concentration ratios as occurs in the mucus of patients with CF in clinical studies with lipoplexes, drastically altered the surface charge and size of our lipoplexes and resulted in the liberation of plasmid DNA from the lipoplexes. These concentration ratios occur in vivo when the DNA concentration in the sputum becomes > 2.7 mg/ml, a quite realistic concentration even in patients without acute exacerbations. Lipoplexes brought in contact with native CF sputa at clinically relevant concentration ratios dissociated when the DNA concentration in the sputa was > 2.7 mg/ml. However, when the linear DNA was degraded by recombinant human deoxyribonuclease I before lipoplexes were added, the linear DNA did not cause any dissociation of the lipoplexes. Addition of albumin and mucin to the lipoplexes in a clinically relevant concentration ratio changed the surface charge of the lipoplexes to negative, however, without release of plasmid DNA. Mucin, dipalmitoylglycerophosphocholine, and dipalmitoylglycerophosphoglycerol did not cause any change in lipoplex properties at clinically relevant concentration ratios.

Albumins↗
PubMed2001

The in vitro evaluation of 'azo containing polysaccharide gels' for colon delivery.

This study reports on 'azo-polysaccharide gels', more specifically azo-inulin and azo-dextran gels, for colon drug delivery. Compared with azo-hydrogels which can be only degraded by reduction of the azo-groups, this study evaluates whether, in vitro, azo-polysaccharide gels can be degraded through both reduction of the azo-groups in the crosslinks as well as enzymatic break down of the polysaccharide backbone. The azo-polysaccharide gels were synthesized by radical crosslinking of a mixture of methacrylated inulin or methacrylated dextran and N,N'-bis(methacryloylamino)azobenzene (B(MA)AB) and were characterized by dynamic mechanical analysis and swelling measurements. Azo-dextran gels could be obtained from methacrylated dextran having low degree of substitution but not from lowly substituted methacrylated inulin. Increasing the amount of B(MA)AB resulted in denser azo-inulin and azo-dextran networks. Compared with their swelling in dimethylformamide, all azo-dextran gels became more swollen in water while azo-inulin gels shrank upon exposure to water, indicating a more hydrophobic character of the azo-inulin gels. Break down of the inulin and dextran chains in the azo-polysaccharide gels by inulinase and dextranase, respectively, was observed. However, the degradation of azo-dextran gels by dextranase seemed to be more pronounced than the degradation of the azo-inulin gels by inulinase. In rat caecal content medium, reduction of the azo function in azo-inulin gels was not observed. This may be attributed to a low partitioning of nicotinamide-adenine dinucleotide phosphate (NADP(+)) in the gels.

Algorithms↗
PubMed2001

An oral controlled release matrix pellet formulation containing nanocrystalline ketoprofen.

A controlled release pellet formulation using a NanoCrystal colloidal dispersion of ketoprofen was developed. In order to be able to process the aqueous NanoCrystal colloidal dispersion into a hydrophobic solid dosage form a spray drying procedure was used. The in vitro dissolution profiles of wax based pellets loaded with nanocrystalline ketoprofen are compared with the profiles of wax based pellets loaded with microcrystalline ketoprofen and of a commercial sustained release ketoprofen formulation. Pellets were produced using a melt pelletisation technique. All pellet formulations were composed of a mixture of microcrystalline wax and starch derivatives. The starch derivatives used were waxy maltodextrin and drum dried corn starch. Varying the concentration of drum dried corn starch increased the release rate of ketoprofen but the ketoprofen recovery remained problematic. To increase the dissolution yield surfactants were utilised. The surfactants were either added during the production process of the NanoCrystal colloidal dispersion (sodium laurylsulphate) or during the pellet manufacturing process (Cremophor RH 40). Both methods resulted in a sustained but complete release of nanocrystalline ketoprofen from the matrix pellet formulations.

Administration, Oral↗
PubMed2001

Biodegradable hydrogels based on stereocomplex formation between lactic acid oligomers grafted to dextran.

A novel hydrogel system in which crosslinking is established by stereocomplex formation between lactic acid oligomers of opposite chirality is proposed. To investigate the feasibility of this novel system, we first investigate whether there is an operation window where lactic acid oligomers in either the D- or L-form do not give a crystalline phase, whereas in a blend of the D- and L-form stereocomplex formation occurs. Therefore, D- and L-lactic acid oligomers with different degrees of polymerization (DP) were prepared and analyzed using DSC. It was shown that crystallinity was present in D- or L-oligomers with DP > or = 11. On the other hand, in blends of D- and L-oligomers of lactic acid crystallinity (stereocomplexation) was already observed at a DP > or = 7. In the next step, L- and D-lactic acid oligomers were coupled via their terminal hydroxyl group to dextran, yielding dex-(L)lactate and dex-(D)lactate, respectively. Upon dissolving each product in water separately and mixing the solutions, a hydrogel is formed at room temperature as demonstrated by rheological measurements. The storage modulus of the obtained hydrogel strongly decreased upon heating to 80 degrees C, while it was restored upon cooling to 20 degrees C demonstrating the thermo-reversibility and the physical nature of the cross-links. The storage modulus of the gels depends on the degree of polymerization of the lactate acid grafts and their degree of substitution on dextran. Interestingly, gel formation was favored when one lactic oligomer was coupled via its hydroxyl group whereas the oligomer of opposite chirality was coupled via its carboxylic acid group. This is ascribed to the parallel packing of the oligomers in stereocomplexes.

Calorimetry, Differential Scanning↗
PubMed2001

Interactions between oligonucleotides and cationic polymers investigated by fluorescence correlation spectroscopy.

PURPOSE: To evaluate whether fluorescence correlation spectroscopy (FCS) can be used to characterize the complexation between oligonucleotides and cationic polymers. METHODS: The features of the complexes between rhodamine labeled oligonucleotides (Rh-ONs) and poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), poly(ethylene glycol)-poly(ethyleneimine) (pEG-pEI), and diaminobutane-dendrimer-(NH2)64 (DAB64) were characterized by light scattering, electrophoretic mobility, electrophoresis, and FCS. RESULTS: At low polymer/Rh-ON ratios, a decrease of the fluorescence of the Rh-ONs was observed on binding of the Rh-ONs to all cationic polymers. This was explained by the creation of "multimolecular complexes" in which the Rh-labels quench each other. The multimolecular complexes, which are highly fluorescent as they carry a number of Rh-ONs, resulted in high fluorescence peaks in the fluorescence fluctuation profile as measured by FCS. For pDMAEMA and DAB64, at higher polymer/Rh-ON ratios the fluorescence of the polyplexes increased, caused by the formation of "monomolecular complexes," which consist of only one Rh-ON per polymer. In the case of pEG-pEI, the fluorescence stayed constant when the polymer/Rh-ON ratio increased, so multimolecular polyplexes remained. FCS confirmed these results as the high fluorescence peaks disappeared in case of pDMAEMA/Rh-ON and DAB64/Rh-ON dispersions, but remained present for pEG-pEI/Rh-ON dispersions. CONCLUSIONS: FCS seems applicable for study of the interactions between ONs and different types of cationic polymers.

Cations↗
PubMed2001

On the release of proteins from degrading dextran methacrylate hydrogels and the correlation with the rheologic properties of the hydrogels.

PURPOSE: To study the release of macromolecules of different sizes (bovine serum albumin, immunoglobulin G) from degrading (addition of dextranase) dextran methacrylate (dex-MA) hydrogels and to correlate the release with the evolution of the rehologic properties of the hydrogels during degradation. METHODS: The size of the macromolecules, the degree of substitution (i.e., number of methacrylates per 100 glycopyranose residues) of the dex-MA and the dextranase concentration in the hydrogels was varied. The rheologic properties were measured with a controlled stress rheometer. RESULTS: The release from dex-MA hydrogels without dextranase was very small [7-20% (time frame up to 180 days)] showing that most of the molecules were entrapped within the hydrogel network. The release from degrading dex-MA hydrogels followed zero-order kinetics for all molecules during a substantial period of the release. This was explained by a liberation and an increasing diffusivity of the proteins in the course of the degradation. The total amount released and the release rates could be well correlated with the rheologically observed degradation rates. CONCLUSIONS: It was shown that rheology can be a useful tool to help explain the release from degrading hydrogels.

Dextranase↗
PubMed2001

Fluorescence fluctuation analysis for the study of interactions between oligonucleotides and polycationic polymers.

The interactions between a cationic polymer, poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh-ON complexes was investigated as a function of the charge ratio (+/-) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/Rh-ON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al, 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several Rh-ONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is connected to the rarity of the fluorescent bursts, which do not provide sufficient data statistics. To specifically address the problem of the fluorescent bursts we employed a method described by Van Craenenbroeck et al. (1999). This method, applicable only when data were integrated over much longer time bins, allowed us to estimate the number of fluorescence bursts which could be considered as a relative measure of the amount of multimolecular complexes present. When monomolecular complexes were formed, i. e. at high values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (epsilon), as revealed from PCH analysis, was greater than epsilon for the free Rh-ONs, indicating that the Rh-ONs were attached to pDMAEMA chains.

Cations↗
PubMed2000

Optimisation of an in vitro procedure for the determination of the enzymatic inhibition potency of multifunctional polymers.

An in vitro procedure for the determination of the inhibition potency of multifunctional polymers towards the proteolytic enzyme trypsin was optimised. Carbopol((R)) 934P was used as the reference polymer. The enzymatic reaction was optimised and the HPLC method was validated. The optimal substrate concentration and enzymatic activity were determined aiming at extracting the linear or steady-state part of the metabolite concentration versus time curve of the enzymatic degradation reaction. A substrate concentration of 20 mmol/l N-alpha-benzoyl-L-arginine-ethylester and an enzymatic activity of 30 enzymatic units trypsin/ml were used. The degree of trypsin inhibition was expressed by the inhibition factor (IF), defined as the ratio of the enzymatic reaction rate without a polymer (control) to the reaction rate in the presence of a polymer. During the optimisation of the trypsin inhibition assay, formation of an ion complex between the substrate and the poly(acrylic acid) was observed. The complex formation was concentration dependent, but the influence on the enzymatic reaction was negligible as long as an excessive substrate concentration was present in the reaction medium. The optimised method allows to characterize, evaluate and compare the in vitro trypsin inhibition strength for most multifunctional polymers.

Acrylic Resins↗
PubMed2000

The physical properties of biogels and their permeability for macromolecular drugs and colloidal drug carriers.

Macromolecular drugs, either free or complexed with colloidal drug carriers, have created a great deal of interest during the last decade. If one wants to administer these new therapeutics via the oral, nasal, and cervical routes or through the conductive airways, one of the first barriers to overcome is the mucus layer that adheres to the related epithelia. In this review, the physicochemical properties of biogels, macromolecular drugs, and colloidal drug carriers that play a major role in transport through biogels are reviewed. Also, methods of studying the mobility of macromolecular drugs and colloidal drug carriers in and through biogels are addressed.

Animals↗
PubMed2000

Pore sizes in hydrated dextran microspheres.

The average pore size of hydrated dextran microspheres is derived from rheological and protein release data. The microspheres were prepared by cross-linking an aqueous solution of methacrylated dextran emulsified in a continuous poly(ethylene glycol) phase. The rheological data were obtained using a novel micromanipulation technique, which enables the compression of a single microsphere. The so obtained pseudoelasticity moduli of the microspheres were derived from these compression data and corresponded well with the elasticity moduli of macroscopic hydrogels of the same composition, as determined with dynamic mechanical analysis. The modulus increased with decreasing water contents of the microspheres and with increasing degrees of methacrylate substitution of the dextran used. Furthermore, the average pore sizes calculated from the pseudoelasticity moduli were in good agreement with the pore sizes derived from protein release data. In conclusion, this study shows that micromanipulation provides insight into the average pore sizes of dextran microspheres, which is an important characteristic that will modulate the release of encapsulated proteins.

Dextrans↗
PubMed2000

Cationic polymer based gene delivery systems.

Gene transfer to humans requires carriers for the plasmid DNA which can efficiently and safely carry the gene into the nucleus of the desired cells. A series of chemically different cationic polymers are currently being investigated for these purposes. Although many cationic polymers indeed condense DNA spontaneously, which is a requirement for gene transfer in most types of cells, the physicochemical and biopharmaceutical behavior of the current generation of polyplexes severely limits an efficient gene transfer in vitro and especially in vivo. This paper summarizes recent physicochemical and biological information on polyplexes and aims to provide new insights with respect to this type of gene delivery system. Firstly, the chemical structure of frequently studied cationic polymers is represented. Secondly, the parameters influencing condensation of DNA by cationic polymers are described. Thirdly, the surface properties, solubility, aggregration behavior, degradation and dissociation of polyplexes are considered. The review ends by describing the in vitro and in vivo gene transfection behavior of polyplexes.

Cations↗
PubMed2000

Cystic fibrosis sputum: a barrier to the transport of nanospheres.

Cystic fibrosis (CF) is characterized by the presence of a viscoelastic mucus layer in the upper airways and bronchi. The underlying problem is a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein. Clinical studies of gene transfer for CF are ongoing. For gene delivery to the airways of CF patients to be effective, the mucus covering the target cells must be overcome. We therefore examined the extent to which CF sputum presents a physical barrier to the transport of nanospheres of a size comparable to that of lipoplexes and other transfection systems currently being clinically evaluated for CF gene therapy. We observed that an extremely low percentage of nanospheres (< 0.3%) moved through a 220-microm-thick CF sputum layer after 150 min. The largest nanospheres studied (560 nm) were almost completely blocked by the sputum, whereas the smaller nanospheres (124 nm) were retarded only by a factor of 1.3 as compared with buffer. Surprisingly, the nanospheres diffused significantly more easily through the more viscoelastic sputum samples. We hypothesize that the structure of the network in sputum becomes more macroporous when the sputum becomes more viscoelastic. Sputum from a patient with chronic obstructive pulmonary disease retarded the transport of nanospheres to the same extent as did CF sputum. When directly mixed with CF sputum, recombinant human deoxyribonuclease I moderately facilitated the transport of nanospheres through CF sputum.

Cystic Fibrosis↗
PubMed1999

Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research.

This review introduces the basics of fluorescence recovery after photobleaching (FRAP) from a theoretical and an instrumentational approach. The most interesting and innovative applications with a pharmaceutical point of view are briefly discussed and possible future applications are suggested. These future applications include research on the mobility of macromolecular drugs in macro- or microscopic pharmaceutical dosage forms, mobility, and binding of antitumor drugs in tumor tissue, intracellular trafficking of gene complexes and mobility of drugs in membranes prior to transmembrane penetration. The paper is also intended to be an introductory guideline to those who would like to get involved in FRAP related experimental techniques. Therefore, comprehensive details on different setups and data analysis are given, as well as a brief outline of the problems that may be encountered when performing FRAP. Overall, this review shows the great potential of FRAP in pharmaceutical research. This is complemented by our own results illustrating the possibility of performing FRAP in microscopic dosage forms (microspheres) using a high resolution variant of FRAP.

Cell Membrane↗
PubMed1998

Characterization of helical cleavages in type II collagen generated by matrixins.

Several vertebrate collagenases have been reported to cleave type II collagen, leading to irreversible tissue destruction in osteoarthritis. We have investigated the action of MMP-1 and MMP-13 on type II collagen by use of neoepitope antibodies and N-terminal sequencing. Previous studies have suggested that the initial cleavage of type II collagen by MMP-13 is followed by a second cleavage, three amino acids carboxy-terminal to the primary cleavage site. We show here that this cleavage is also produced by APMA-activated MMP-1 in combination with MMP-3 (i.e. fully activated MMP-1). The use of a selective inhibitor of MMP-3 has shown that it is this enzyme, rather than interstitial collagenase which had been exposed to MMP-3, which makes the second cleavage. In addition we have identified, through N-terminal sequencing, a third cleavage site, three residues carboxy-terminal to the secondary site. Since MMP-2 is thought to be responsible for gelatinolytic action on type II collagen we have investigated the effect of MMP-2 after the initial helical cleavage made by either MMP-1 or MMP-13. A combination of MMPs-1, -2 and -3 results in both the second and third cleavage sites; adding MMP-2 to MMP-13 did not alter the cleavage pattern produced by MMP-13 on its own. We conclude that none of the three cleavage sites will provide information about the specific identity of the collagenolytic enzymes involved in collagen cleavage in situ. Staining of cartilage sections of osteoarthritis patients with the neoepitope antibodies revealed type II collagen degradation starting at or near the articular surface and extending into the mid and deep zones with increasing degeneration of the cartilage.

Animals↗
PubMed1996

New international F.I.P. method for the determination of the activity of Aspergillus oryzae proteases.

Proteases of Aspergillus oryzae are used as a drug in the therapy of digestive disorders. To standardize these enzyme the Enzyme Commission of the Fédération Internationale Pharmaceutique (F.I.P.) has tested a new determination method, which will be described below. The standard preparation of a mixture of Aspergillus oryzae proteases used in this test is characterized.

Aspergillus oryzae↗
PubMed1995

Kinetic constants for the hydrolysis of aggrecan by the papaya proteinases and their relevance for chemonucleolysis.

The four known proteinases from papaya latex, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl endopeptidase (EC 3.4.22.25), were purified to homogeneity and fully characterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kinetic constants for aggrecan hydrolysis by the papaya proteinases. The disappearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the Km was much larger than 0.5 microM and that kcat/Km = 1.2 +/- 0.1 x 10(6) M-1 s-1 for chymopapain, 1.20 +/- 0.08 x 10(6) M-1 s-1 for caricain, 0.90 +/- 0.02 x 10(6) M-1 s-1 for papain, and 0.120 +/- 0.005 x 10(6) M-1 s-1 for glycyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggrecan hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observations, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis.

Aggrecans↗
PubMed1994

Fractionation and purification of the thiol proteinases from papaya latex.

Three cysteine proteinases, i.e. chymopapain, papaya proteinase IV and proteinase III, were purified to homogeneity from papaya latex using a combination of ion-exchange chromatography and hydrophobic interaction chromatography. During the purification procedure, the thiol-groups of the active center were reversibly blocked as mixed disulfides with 2-thiopyridone. Homogeneity was proved electrophoretically by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)-PAGE and rechromatography on a Mono S 5/5 column at pH 5.0.

Amidohydrolases↗