Biomedical subjects

M Baudys

Publications and source records attributed to M Baudys.

At least 19 recordsLinked to original sources

PubMed2001

In situ study of insulin aggregation induced by water-organic solvent interface.

PURPOSE: The aim of this study was to assess insulin stability by monitoring in situ time-course of insulin aggregation induced by a water-organic solvent (o/w) interface that occurs during the microencapsulation process. METHODS: Insulin aggregation at a simple o/w interface was monitored spectrophotometrically by detecting the percentage of turbidity changes (%T) at 350 nm. The effects of protein concentration and agitation and the presence of poly (lactic-co-glycolic acid) (PLGA) in methylene chloride (MC) on insulin aggregation were observed. For the 0.72 mg/ml insulin in phosphate-buffered saline (PBS), the effect of nonionic (dodecyl maltoside [DDM]) and anionic (sodium dodecyl sulfate [SDS]) surfactant in PBS were also evaluated at various protein/surfactant mol ratios. The conformation of insulin protected by a 10-fold molar excess of SDS recovered after 1 h of contact with MC was evaluated via circular dichroism (CD) spectroscopy. RESULTS: A typical turbidity-time profile was represented by a sigmoidal curve. Greater change in %T was observed with increasing insulin concentration, in the presence of PLGA in MC and in the presence of agitation. DDM failed to delay insulin aggregation at all ratios used, whereas a less than 10% change in %T was observed in 1 h when a 10- - approximately 20-fold excess of SDS was used. CD spectra indicated that the presence of insulin in SDS after 1 h of contact with MC qualitatively retained its secondary structure integrity. CONCLUSIONS: An experimental method was designed for an in situ assessment of protein stability at the o/w interface.

Chemical Phenomena↗

PubMed2001

The effect of zinc-crystallized glucagon-like peptide-1 on insulin secretion of macroencapsulated pancreatic islets.

This research investigates the use of an insulinotropic factor, glucagon-like peptide-1 (GLP-1), to enhance insulin secretion from islets within a macrocapsule. A zinc-crystallized form of GLP-1 was added to the macrocapsule device to have a longer and more controlled release of the bioactive monomer GLP-1. The type of macrocapsule device used for this study consisted of a hollow fiber (MWCO 100,000 and 1 mm inner diameter) containing rat islets and GLP-1 crystals within a poly(N-isopropylacrylamide-co-acrylic acid) (2 mol% acrylic acid) matrix. When incubating the system in media with a high glucose concentration (300 mg/dL), insulin secretion was enhanced with a >85% increase after an induction period. When the same type of system was used in a dynamic perfusion experiment, similar results were obtained. GLP-1 crystals can be an effective form to be entrapped in a bioartificial pancreas to enhance insulin secretion function, especially at high glucose concentrations.

Animals↗

PubMed2000

Arginine-based structures are specific inhibitors of cathepsin C. Application of peptide combinatorial libraries.

Novel synthetic peptide inhibitors of lysosomal cysteine proteinase cathepsin C have been designed through the use of soluble peptide combinatorial libraries. The uncovered structural inhibitory module consists of the N-terminal cluster of L-arginine residues. Its modification with D-amino acids or arginine derivatives did not increase the inhibition strength. Inhibitory potency of oligoarginines improves with the elongation of peptide chain reaching a maximum for octa-L-arginine. The oligoarginines specifically interact with the cathepsin C active site as shown by competitive-type inhibition kinetics (Ki approximately 10-5 M) and intrinsic fluorescence measurements. The inhibitory interaction of oligoarginines is established through the specific spatial contact of a net of guanidino groups in the arginine side-chains, as indicated by comparison with inhibitory action of low molecular mass guanidine derivatives (Ki approximately 10-3 M). Nonarginine polyionic compounds cannot mimic the inhibitory effect of oligoarginines. The arginine-based peptide inhibitors were selective towards cathepsin C among other cysteine proteinases tested.

Animals↗

PubMed2000

Effect of ionic strength on the loading efficiency of model polypeptide/protein drugs in pH-/temperature-sensitive polymers.

In this report, the effect of ionic strength on the loading efficiency of three model polypeptide/protein drugs, namely angiotensin II, insulin, and cytochrome c, in pH- and temperature-sensitive terpolymers of poly(NIPAAm-co-butylmethacrylate-co-acrylic acid) (poly(NIPAAm-co-BMA-co-AA)) has been investigated. Loading efficiency of polypeptides in pH-/temperature-sensitive beads composed of poly(NIPAAm-co-BMA-co-AA) terpolymer is predominantly governed by hydrophobic interactions, both nonspecific surface interactions and/or specific interactions (binding pockets) between the protein and the polymer molecules. Thus, loading efficiency increases with ionic strength. However, as ionic strength increases further, polymer deswelling (collapse), which is also controlled by hydrophobic forces, becomes more pronounced, and consequently, a higher fraction of water is squeezed out during bead formation and the loading efficiency starts to decrease.

Acrylamides↗

PubMed1999

Modulating insulin-release profile from pH/thermosensitive polymeric beads through polymer molecular weight.

Stimuli-sensitive statistical terpolymers of N-isopropylacrylamide (NIPAAm) (temperature-sensitive), butyl methacrylate (BMA) and acrylic acid (AA) (pH-sensitive) of various molecular weight (MW) with NIPAAm/BMA/AA feed mol ratio of 85/5/10 were used to modulate release of insulin, a model protein drug, from pH/thermosensitive polymeric beads. Protein drug loading from an aqueous medium into the beads was achieved by preparing a 7 or 10% (w/v) polymer solution with 0.2% (w/v) insulin at low pH and below the lower critical solution temperature (LCST) of the polymer (pH 2.0 and 4 degrees C), and then dropping the solution into an oil bath above the LCST of the solution (35 degrees C). This loading procedure maintained protein stability while achieving high loading efficiency, between 90 and 95% in the beads. Insulin-release studies from beads prepared from terpolymers of the same composition but increasing MW were performed at pH 2.0 and 7.4, at 37 degrees C. It was observed that there was negligible loss of insulin at pH 2.0 from the beads, indicating no burst effect. At pH 7.4, insulin release was seen from all the beads and the release rate was a function of the MW of the polymer. The low MW polymeric beads eroded, dissolved and released most of the insulin within 2 h at pH 7.4 and 37 degrees C, the intermediate MW polymeric beads swelled slightly, dissolved and released most of the insulin within 4 h, whereas the high MW polymeric beads swelled slowly and gradually released the loaded insulin over a period of 8 h. Thus, the release of protein from the low MW polymeric beads is controlled by the rate of dissolution of the polymer, whereas the release from the high MW polymeric beads is controlled by swelling of the beads and drug diffusion. Studies using fluorescein-labeled insulin revealed that insulin was uniformly distributed in the beads regardless of polymer MW. The loaded and released insulin were fully bioactive. Based on the described results, the low MW polymeric beads may be used for immediate delivery of protein drugs in the duodenum, the intermediate MW polymeric beads may be used for lower small intestine targeting, while the high MW polymeric beads may be used to target protein drugs predominantly to the colon.

Administration, Oral↗

PubMed1999

Site-specific insulin conjugates with enhanced stability and extended action profile.

Two different hydrophilic moieties, carboxyl derivatives of monosaccharidic (Glc, Gal, Man, Fuc) glycosides and methoxypolyethylene glycols of varying MW, were covalently attached to the insulin GlyA1, PheB1 and/or LysB29 amino groups (seven possible derivatives), and resulting insulin conjugates purified to homogeneity. In vivo bioactivity in rats of most derivatives was preserved while disubstituted PEG-insulins showed decreased potency. Only site-specific modification of PheB1 amino group with either moiety resulted in pronouncedly increased resistance of insulin to fibrillation, indicating that the B-chain N-terminus of the insulin molecule is mechanistically involved in the fibrillation process. Immunogenicity in vivo and in vitro of monoglycosylated insulins was comparable to that of insulin, diglycosylated insulins showed immunogenicity enhancement. Immunogenicity of PEG-insulins was significantly suppressed. PheB1-glycosylated insulins administered subcutaneously in dogs displayed extended action profiles, the most effective being PheB1-galactosylated insulin, resembling the pharmacodynamic response of intermediate-acting insulin preparations. The pharmacokinetic parameters of these insulin derivatives were not significantly different from that of insulin even though absorption and residence time and clearance were increased, providing some explanation for prolonged action profile. Lectin-specific binding as a retardation basis is not likely involved. In support of this, subcutaneously administered PheB1-PEG(600)-insulin showed an even more protracted action profile, suggesting that the basis of retardation is physical and nonspecific. This implies that by increasing PEG chain MW, further delay/prolongation of action can be achieved to yield new soluble basal insulin substitutes with potential clinical applications.

Journal Article↗

PubMed1999

Polymer molecular weight alters properties of pH-/temperature-sensitive polymeric beads.

PURPOSE: To study the physical and release properties of different molecular weight (MW) pH- and temperature-responsive statistical terpolymers and beads of N-isopropylacrylamide (NIPAAm), butylmethacrylate (BMA) and acrylic acid (AA). METHODS: Random terpolymers of varying MW were synthesized with NIPAAm/BMA/AA of feed mol ratio 85/5/10. Polymeric beads were formed by dropping a polymer solution into an oil bath kept at a temperature above the lower critical solution temperature (LCST) of the polymer. The release profile of cytochrome c was investigated as a function of the polymer MW and pH of the release medium at 37 degrees C. RESULTS: The weight average MW ranged between 49,000 and 3 million. The LCST at pH 2.0 and pH 7.4 was 22 degrees C and 60 degrees C respectively. SEM studies showed that the size of the pores decreased as the MW increased. Irrespective of MW, the polymeric beads did not swell or dissolve at pH 2.0 and 37 degrees C and showed minimal drug release. At pH 7.4 and 37 degrees C, the rate of bead dissolution/swelling decreased as the MW of the polymer increased. CONCLUSIONS: By modulating polymer MW, it was possible to vary the physical properties of beads. Dissolution/swelling characteristics were dependent on the MW of the polymer. Such unique dissolution/swelling properties are useful for delivering drugs to different sites in the intestine.

Acrylates↗

PubMed1999

Effect of molecular weight and polydispersity on kinetics of dissolution and release from ph/temperature-sensitive polymers.

N-isopropylacrylamide (NIPAAm) polymers exhibit a lower critical solution temperature (LCST). Aqueous solutions of these polymers are soluble below their LCST and precipitate above their LCST. The LCST is dependent on pH for polymers with ionizable groups because of a change in hydrophilicity with ionization and electrostatic repulsion that cause a shift in the LCST. We have designed a novel polymeric delivery system that utilizes linear, pH/temperature-sensitive terpolymers of NIPAAm, butyl methacrylate (BMA) and acrylic acid (AA). This system allows the aqueous loading of drugs in polymeric beads with high loading efficiency while preserving the bioactivity of the protein drug. Furthermore, the unique properties of the pH/temperature-sensitive polymeric bead make it a potential system for oral drug delivery of peptide and protein drugs to different regions of the intestinal tract. This study aims at investigating the effect of polydispersity and molecular weight (MW) of terpolymers of poly(NIPAAm-co-BMA-co-AA) with feed mol ratio of NIPAAm/BMA/AA 85/5/10 on the polymer dissolution rate and on the release kinetics of a model protein, namely insulin. Varying the weight average MW (Mw) and polydispersity of the polymer modulated the polymer dissolution rate and the release rate of insulin from pH/temperature-sensitive polymeric beads. An increase in the polydispersity of the polymer through the addition of high MW polymer chains caused a decrease in the release rate of insulin and in the polymer dissolution rate. High MW polymer chains impose a certain degree of interaction between polymer chains due to chain entanglement. There is a limiting value of MW above which chain entanglement has no effect on drug release rate.

Acrylamides↗

PubMed1998

Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs: complexation and stabilization of insulin.

Insulin (Ins) spontaneously and easily complexed with the hydrogel nanoparticle of hydrophobized cholesterol-bearing pullulan (CHP) in water. The complexed nanoparticles (diameter 20-30 nm) thus obtained formed a very stable colloid. The thermal denaturation and subsequent aggregation of Ins were effectively suppressed upon complexation. The complexed Ins was significantly protected from enzymatic degradation. Spontaneous dissociation of Ins from the complex was barely observed, except in the presence of bovine serum albumin. The original physiological activity of complexed Ins was preserved in vivo after i.v. injection.

Animals↗

PubMed1997

The in vivo antibody response against exogenous antigens is not influenced by the mouse Bcg (Nramp1) gene.

The mouse Nramp1 (Bcg) gene on chromosome 1 exerts pleiotropic effects on macrophage function. The gene is known to affect presentation of mycobacteria, and other antigens in vitro, so that macrophages carrying the resistant Bcg allele better support the proliferation of antigen-specific T cells compared with macrophages of the sensitive phenotype. To determine whether the Bcg allele could affect in vivo the antibody response to antigens not related to mycobacterial infections, we tested the primary and secondary responses to sheep red blood cells (SRBC) and glycosylated bovine insulin (G-insulin) in two pairs of Bcg congenic strains: BALB/c (Bcgs) versus BALB/c.CD2 (Bcgr), and B10.A (Bcgs) versus B10Ar (Bcgr), and in C57BL/10ScSn (B10; Bcgs) and A/J (Bcgr) mice. Furthermore, the antigen-specific proliferative responses of T cells primed in vivo by protein antigens were also tested in Bcg congenic mice. We found no significant difference in in vivo antibody response either to SRBC or G-insulin between the Bcgr and Bcgs strains. The magnitude of in vitro antigen-specific proliferation of lymph node cells sensitized in vivo by hen egg lysozyme (HEL) or chicken ovalbumin (OVA) was also similar in Bcgs and Bcgr congenic mice. However, we have documented a higher antigen-presenting capacity of Bcgr macrophages in in vitro antigen-specific proliferation to OVA. Since the macrophages are the only cells in which the Nramp1 gene is expressed, we suggest that the activity of other types of antigen-presenting cells masks the effect of the Bcgr allele on antigen-presentation in vivo.

Animals↗

PubMed1996

Conjugates of insulin with copolymers of N-(2-hydroxypropyl) methacrylamide: effects on smooth muscle cell proliferation.

The hypothesis that an elevated plasma insulin level contributes to an increase in coronary heart disease has led to studies of the mitogenic effect of native insulin and its conjugates on smooth muscle cells (SMC). In this study, insulin was covalently attached to two water-soluble polymers containing N-(2-hydroxypropyl)methacrylamide using the mixed anhydride method. The first polymer was a copolymer of N-(2-hydroxypropyl)methacrylamide and N-methacryloyldiglycine. The second one was a terpolymer of two of the above-given monomers and R-(-)-1-methyl-2-methacryloylamidoethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. Insulin conjugates were isolated and characterized, and the mitogenic effect on SMC was investigated. The results showed that only conjugates of insulin and terpolymers bearing pendant N-acetyl-glucosamine groups do not have a mitogenic effect on SMC while maintaining the hypoglycemic activity of insulin. This finding suggests that some inter- or intramolecular interactions of coupled insulin with the sugar moiety(ies) attached to the polymer backbone contribute to the observed effects.

Acrylic Resins↗

PubMed1996

Temperature and pH-sensitive polymers for human calcitonin delivery.

PURPOSE: Stimuli-sensitive polymers are suitable candidates for oral peptide drug delivery vehicles since they will prevent gastric degradation in the stomach while providing a controlled release of a peptide drug such as calcitonin later. The purpose of this study was to fabricate polymeric beads from pH/temperature sensitive linear terpolymers (poly(N-isopropylacrylamide-co-butylmethacrylate-co-acrylic acid) and load them with a peptide drug, human calcitonin, which was dissolved in aqueous phase. METHODS: The polymeric beads were formed by solubilizing a cold, aqueous solution of temperature sensitive polymer with human calcitonin. This solution was added dropwise into an oil bath kept at a temperature above the LCST of a polymer, precipitating polymer and entrapping the peptide. The quantity and the physical state of the peptide were analyzed by reverse-phase HPLC, CD and FTIR and its biological activity after loading was determined in vivo. RESULTS: The loading efficiency and stability of human calcitonin into the polymeric beads was studied as a function of pH and ionic strength of the loading buffer and temperature of the oil bath. Final optimal loading conditions were 20 mM glycine/HCl buffer, pH 3.0 containing 0.15 M NaCl as a dissolution medium and 23 degrees C as the oil bath temperature. Loading and release of human calcitonin were also studied as a function of acrylic acid content in the terpolymers. As the acrylic acid content increased from 0 to 10 mol %, the loading efficiency and stability of calcitonin improved significantly. The same trend was observed for the quantity of released calcitonin. In vivo biological activity of the released hormone was preserved. CONCLUSIONS: The results showed that the beads made of the polymers with high content of acrylic acid (most hydrophilic) provided better loading, stability and release of human calcitonin. The designed beads represent a new potential system for oral delivery of calcitonin and other peptides.

Acrylamides↗

PubMed1995

Physical stabilization of insulin by glycosylation.

The modification of human insulin by the covalent attachment of monosaccharide moieties to insulin amino group(s) alters the aggregation and self-association behavior, improving both the pharmaceutical stability and biological response. The synthesis of p-succinamidophenyl glucopyranoside-insulin conjugate(s) (SAPG-insulin) has resulted in seven possible glucosylated insulin derivatives (three monosubstituted, three disubstituted, and one trisubstituted). These derivatives were isolated and purified using ion exchange chromatography. Characterization of the derivatives includes determining the site and number of sugar groups attached for each individual derivative and an evaluation of biological activity. Nearly all the derivatives retained in vivo biological activity comparable to insulin. In addition, extensive physicochemical characterization of the glucosylated insulin derivatives was undertaken to determine association/aggregation properties using GPC, dynamic light scattering, UV/Vis, and CD spectroscopy. Protein self-association was most suppressed with the disubstituted derivatives, especially those substituted on PheB1, and the trisubstituted derivative. The same general pattern was observed for physical stability of glucosylated insulin derivatives. As the number of glucosyl moieties attached to insulin increased, solution physical stability dramatically improved. Yet, the most significant impact to stability was glycosylation at the PheB1 site.

Amino Acid Sequence↗

PubMed1993

Human breast milk contains procathepsin D--detection by specific antibodies.

The presence of the zymogen of cathepsin D in human milk was detected using antibodies specific for the proenzyme and by the proteolytic activity at low pH. The antibodies were raised against a synthetic propeptide of human cathepsin D and were tested using immunoprecipitations and western blots of samples from different breast cancer cell lines as well as cytosol fractions of human breast cancer tissues. In all experiments these antibodies recognized specifically procathepsin D. Procathepsin D from human milk was partially activated at low pH. The activity was monitored using hemoglobin 14C proteolytic assay, and it was abolished by pepstatin A--a specific inhibitor of aspartic proteinases. Western blots did not reveal presence of cathepsin B or cathepsin H. These data indicate specific secretion of cathepsin D in human breast milk.

Antibodies↗

PubMed1992

Purification and crystallization of human cathepsin D.

The two-chain form of human cathepsin D was purified from human spleen with a method utilizing an ion exchange chromatography step prior to the pepstatin affinity column normally used to purify aspartic proteases. The protein was crystallized from 21% polyethylene glycol 8000 at pH 4.0 using the hanging drop vapour diffusion method. Small crystals were used as seeds to grow crystals suitable for X-ray data collection. The crystals diffract to a resolution of 3.2 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 59.9 A, b = 99.6 A, c = 133.6 A. There are two molecules in the asymmetric unit.

Cathepsin D↗

PubMed1992

New trypsin inhibitors are present in the coelomic fluid of the earthworm Lumbricus terrestris.

Proteinase-inhibiting components of the coelomic fluid of the earthworm Lumbricus terrestris were examined. Inhibition of proteinases of serine, aspartate and thiol families was tested. Very strong inhibition was observed only in the case of trypsin. Additional data suggest that the inhibition is related to proteins of molar mass of 42 kDa and 20 kDa, respectively. These two proteins are present in the coelomic fluid in several forms which differ in their isoelectric points.

Animals↗

PubMed1991

Inhibition of aspartic proteinases by propart peptides of human procathepsin D and chicken pepsinogen.

Two propart peptides of aspartic proteinases, the propart peptide of chicken pepsin and human cathepsin D, respectively, were investigated from the point of view of their inhibitory activity for a set of aspartic proteinases. These peptides display a very broad inhibitory spectrum. The strongest inhibition was observed for pepsin A-like proteinases where propart peptides can be used as titrants of active enzymes.

Animals↗

PubMed1991

Crystallization and preliminary crystallographic study of cathepsin D inhibitor from potatoes.

Single crystals of the glycosylated inhibitor of cathepsin D and trypsin isolated from potato tubers were obtained using the hanging drop vapor diffusion method and ammonium nitrate as precipitant. The crystals exhibit strong F222 pseudo symmetry but belong to the orthorhombic space group C222 or C222(1), with cell parameters a = 73.8 A, b = 119.9 A and c = 133.2 A with two molecules per asymmetric unit. The crystals diffract to a resolution of 2.4 A.

Cathepsin D↗