Polymethyacrylate based microparticulates of insulin for oral delivery: preparation and in vitro dissolution stability in the presence of enzyme inhibitors

The purpose of this investigation was to (a) evaluate the coprecipitation technique for preparing microparticulates of insulin, (b) study the effect of variables such as addition of salts in the precipitating medium and ratio of polymeric solution to volume of precipitating medium on the dissolution and encapsulation efficiency of insulin microparticulates, and (c) evaluate the in-vitro enzymatic dissolution stability of insulin microparticulates in the presence of chicken ovomucoid (CkOVM) and duck ovomucoid (DkOVM) as inhibitors. Insulin dissolved in 0.01 N HCl was mixed with alcohol USP to get a final concentration of 32% v/v. Eudragit L100, a representative polymethyacrylate polymer, was then dissolved in this solution which was transferred to a beaker containing cold water with homogenization to obtain microparticulates. Dissolution studies were carried out in pH 6.8 phosphate buffer using a 100-ml conversion kit in a standard dissolution assembly. Dissolution stability of microparticulates was evaluated in the presence of 0.5 microM trypsin and 0.l microM chymotrypsin at various ratios of CkOVM and DkOVM. The results indicated that insulin microparticulates could be prepared using the coprecipitation technique with high encapsulation efficiency by proper selection of experimental conditions and amount of polymer. Presence of salts in the precipitating medium decreased the dissolution of insulin from the microparticulates. As the ratio of precipitating medium with respect to the polymeric solution was increased, the encapsulation efficiency increased. In dissolution stability experiments, insulin was not detected in the presence of enzymes alone. When CkOVM and DkOVM were incorporated, the stability of insulin increased significantly in a concentration dependent fashion.     

Protection of salmon calcitonin breakdown with serine proteases by various ovomucoid species for oral drug delivery

The current work compared protective effects of various ovomucoid species against salmon calcitonin (sCT) metabolism by serine proteases. sCT solutions (50 microM) were incubated at 37 degrees C with trypsin (0.5 microM), alpha-chymotrypsin (0.1 microM), or elastase (0.48 microM) in 50 mM Tris buffer (pH 8.0) containing or lacking different concentrations of turkey ovomucoid (tOVM), duck ovomucoid (dOVM), or chicken ovomucoid (cOVM) and aprotinin. Caco-2 cell homogenate was also incubated with sCT and the contents of the proteases were assayed by using their specific substrates. Metabolites were identified by high-performance liquid chromatography, gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry techniques. In the absence of inhibitors, there was a considerable degradation of sCT by the proteases. dOVM and tOVM increased the half-life of sCT with trypsin and alpha-chymotrypsin at enzyme-to-inhibitor ratio of 1:4 showing similar efficacy. dOVM was found to be superior to tOVM in protecting sCT from elastase. cOVM was ineffective in protecting sCT against alpha-chymotrypsin. Caco-2 cell homogenate degraded sCT, which was prevented by tOVM. sCT was cleaved into different molecular weight fragments with different proteases. In general, the metabolite formation decreased when inhibitor concentration increased. dOVM and tOVM effectively stabilized sCT against all three proteases. However, cOVM could not prevent the degradation by alpha-chymotrypsin.     

INTRAMOLECULAR CROSS-LINKING OF PROTEINS BY FORMATION OF LYSINOALANINE OR LANTHIONINE Modification of Disulfides in Ovomucoids

The disulfide bonds of ovomucoids were cleaved and new sulfur-containing cross-links were introduced by two separate chemical modification methods: (1) alkali treatment, and (2) cyanolysis. Alkali and cyanolytic treatments were used to cleave disulfide bonds and to introduce new synthetic nonreducible cross-links consisting of residues of lysinoalanine (N-(dl -2-amino-2-carboxyethyl)-l -lysine) and lanthionine (bis(2-amino-2-carboxyethyl)sulfide). The two ovomucoids studied were turkey and penguin ovomucoids, which are “double-headed” inhibitors of proteolytic enzymes with one inhibitory site for bovine trypsin and another for bovine α-chymotrypsin or subtilisin. Trypsin does not compete with either α-chymotrypsin or subtilisin, but the latter compete with one another, apparently for the same inhibitory site. The stability of inhibitory activities as a function of disulfide bond scission and the formation of new nondisulfide cross-links was studied. In both methods of disulfide modification of turkey ovomucoid, inhibitory activity against trypsin was more stable with respect to the extent of modification than was inhibitory activity against chymotrypsin or subtilisin. With penguin ovomucoid, inhibitory activity against subtilisin was always more stable than activity against trypsin or chymotrypsin with both methods. With both ovomucoids it was thus possible to produce single-headed inhibitors from the double-headed inhibitors. The formation of the nonreducible cross-links of lanthionine from cystines, and of lysinoalanines from cystines and lysines, with retention of a biochemical activity, suggests that such a procedure may have at least limited use as a cross-linking method.     

Transdermal delivery of antiparkinsonian agent, benztropine. I. Effect of vehicles on skin permeation

The influence of pH and various lipophilic and hydrophilic vehicles on the epidermal permeation of benztropine (BZ) free base and its mesylate salt were studied in vitro using the hairless mouse (HLM) and human cadaver (HC) skin membranes. The pH-partition behavior of BZ base (pK(a)=10) was examined using n-octanol and Britton-Robinson buffers over the pH range of 5-12. Unexpectedly, the ionized species of BZ yielded a high partition coefficient (log K(octanol/water)=2. 14), which was reflected by its relatively high skin permeability (P=1.6x10(-2)cm h(-1)). BZ base delivered from a lipophilic vehicle with a solubility parameter range of 7.1-10.3 (cal cm(3))(1/2) exhibited a significantly enhanced rate of permeation as compared to that attained from a hydrophilic vehicle of solubility parameter range between 12.5-23.4 (cal cm(3))(1/2). Among the neat solvents examined, a lipophilic carrier, isopropyl myristate (IPM) provided the most enhancing effect on the permeation of BZ base. In addition, the neat IPM carrier offered the maximum BZ base flux of 150 microg per cm(2) h(-1) across HC skin, which was approximately 16 times greater than the target delivery rate of BZ from a 10-cm(2) device. In comparison, BZ base exhibited a 2-60 times greater flux than BZ mesylate when delivered from the neat solvents. However, interestingly enough, the binary cosolvents consisting of IPM and short-chain alkanols such as ethanol (EtOH), isopropanol (iPrOH), and tertiary butanol (tBtOH), in particular a 2:8 combination, produced a marked synergistic enhancement of BZ flux from the mesylate salt, whereas a retarding effect was noticed for the permeation of BZ base. The enhancement potency for the BZ mesylate permeation increased linearly with the carbon number of the branched alcohols present in the binary mixtures. A tBtOH-IPM (2:8) combination produced the highest BZ flux from the mesylate salt, i.e. , 2016 mg per cm(2) h(-1), which was 100-fold greater than from water and 44-540-fold greater than the individual neat solvents, respectively. The observed permeation enhancement of BZ mesylate by the alkanol-IPM mixtures was probably as a result of a combination of decreasing barrier ability of the stratum corneum by the binary vehicles and moderately partitioning BZ mesylate through the viable epidermis/dermis.     

Intraocular distribution of topically applied hydrophilic and lipophilic substances in rat eyes

Purpose: Topical administration is the preferred route of drug delivery for ophthalmic ailments. However, poor permeation through ocular surface and significant systemic absorption, makes the topical drug delivery challenging. Furthermore, distribution of topically delivered drugs varies with their physicochemical properties and the type of formulation used. Hence, this study was done to understand the pattern of ocular drug distribution of topically applied hydrophilic and lipophilic substances in two different formulations.

Methods: 5-Carboxyfluorescein and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate were used as representative candidates for hydrophilic and lipophilic substances, respectively. They were formulated in solution and liposomes. Single drop of either formulation containing hydrophilic or lipophilic substance was instilled topically, unilaterally to rat eyes. Subsequently, rats were sacrificed at 10, 30 and 120?min post-instillation. Eyes were cryosectioned and examined under confocal microscope to determine the fluorescence intensity in ocular tissues.

Results: Corneal permeation of hydrophilic and lipophilic substances in both formulations peaked at 30?min post-instillation. Liposomal–lipophilic dye and non-liposomal–hydrophilic dye showed better corneal distribution. Fluorescence was absent in contralateral eyes of non-liposomal–hydrophilic dye-treated animals but was present in contralateral eyes of liposomal–hydrophilic dye-treated animals. Fluorescence in contralateral eyes of liposomal–lipophilic dye-treated animals was significantly higher compared to non-liposomal–lipophilic dye-treated animals.

Conclusions: Topically applied liposomal formulation of lipophilic substance provides higher corneal concentration of drug with lesser systemic absorption compared to its solution. For hydrophilic substance, topical use of solution provides greater corneal concentration compared to liposomes which is more likely to be absorbed systemically.     

Computer Simulation of Skin Permeability of Hydrophobic and Hydrophilic Chemicals – Influence of Follicular Pathway

A recently published mechanistic skin permeability model (Kasting et al., 2019. J Pharm Sci 108:337-349) that included a follicular diffusion pathway has been extended to describe transient diffusion and finite dose applications. The model follows the disposition of two components, solute and solvent, so that solvent deposition processes can be explicitly represented. Experimentally-calibrated permeability characteristics of the follicular pathway leading to the permeation of highly hydrophilic permeants are further refined. Details of the refinements and a comparison with the earlier model using two large experimental datasets are presented. An example calculation shows the marked difference between the time scales for achievement of near steady-state diffusion for large hydrophilic and lipophilic compounds, with the former being more than 100-fold faster than the latter. However, the true steady state for the hydrophilic compound is not reached until much later due to the very slow filling of the corneocyte phase.     

The effect of various in vitro conditions on the permeability characteristics of the buccal mucosa

The effect of various in vitro conditions on the permeability characteristics of the buccal mucosa was assessed using caffeine (CAF) and estradiol (E(2)) as model hydrophilic and lipophilic markers, respectively. The permeation of CAF and E(2) through porcine buccal mucosa was determined in modified Ussing chambers at 37 degrees C over 4 h. Comparative permeation studies were performed through full thickness and epithelial tissues, fresh and frozen tissues, and intact and intentionally damaged tissues. Tissue integrity was monitored by the absorption of the normally impermeable fluorescein isothiocyanate (FITC)-labeled dextran 20 kDa (FD20) and tissue viability was assessed using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) biochemical assay and histological evaluation. Compared to full thickness buccal tissue, permeability through the buccal epithelium was 1.8-fold greater for CAF and 16.7-fold greater for E(2). Although the fluxes of the model compounds were no different in fresh and frozen buccal epithelium, histological evaluation demonstrated signs of cellular death in frozen tissue. FD20 permeated damaged tissue, and while this was associated with an increase in CAF transport, no significant change in E(2) transport was observed. The tissue appeared to remain viable for up to 12 h postmortem using the MTT viability assay, and this was supported by histological evaluation.     

A simple HPLC method for the simultaneous analysis of insulin and ovomucoid

An analytical HPLC method is reported for the simultaneous determination of insulin and its enzyme inhibitor, chicken ovomucoid. Verapamil was used as an internal standard. The elution was achieved using a gradient technique (10-15% B for 4 min, 15-35% B from 5th to 11th min and 35-10% B from 12th to 22nd min). The mobile phase used was 0.05% v/v trifluoroacetic acid (TFA) in water and 0.05% v/v TFA in acetonitrile with a flow rate of 1.2 ml/min. The analytes were detected at 210 nm after resolution using a reversed phase C-18 column. Insulin, ovomucoid and verapamil (IS) were eluted at 11.9, 14.2, and 18 min, respectively, free from any interfering endogenous peaks during a run time of 22 min. Linear relationships were observed between the detector response and the concentrations of the analytes (0.05-1 I.U/ml for insulin (r2 = 0.9975) and 5-100 microg/ml for the chicken ovomucoid (r2 = 0.9993)). The assay was found to be highly selective and sensitive due to the absence of any interfering peaks. The lower C.V and % error values of the assay indicates that the assay could accurately and precisely quantitate both insulin and ovomucoid in the examined concentration range. This method can be used for the simultaneous quantitation of insulin and chicken ovomucoid.     

Influence of phloretin on the skin permeation of lidocaine from semisolid preparations.

The purpose of the present study was to determine the significance of phloretin on the topical permeation of lidocaine using different semisolid preparations as delivery systems. One hydrophilic and three lipophilic formulations were used. After estimation of the solubility of phloretin and lidocaine in the vehicles and analysis of the viscoelastic properties, standard diffusion experiments with Franz type diffusion cells through porcine skin were performed. Results indicate a general lidocaine enhancement by phloretin in the tested vehicles. The permeation was enhanced 1.39-fold in the hydrophilic formulation and between 1.25- and 1.76-fold in the lipophilic formulations.     

Improvement of subcutaneous bioavailability of insulin by sulphobutyl ether beta-cyclodextrin in rats

The objective of this study was to examine and compare how hydrophilic beta-cyclodextrin derivatives (beta-CyDs) improve the bioavailability of insulin following subcutaneous injection of insulin solution in rats. When insulin solutions in the absence of beta-CyDs were injected into the dorsal subcutaneous tissues of rats, the absolute bioavailability of insulin calculated from plasma immunoreactive insulin (IRI) levels was approximately 50%. When maltosyl-beta-cyclodextrin was added to the solutions, there was no change in the plasma IRI levels and hypoglycaemia compared with those of the insulin-alone solution. Dimethyl-beta-cyclodextrin decreased the bioavailability of insulin, although it increased the maximal concentration of IRI in plasma and the capillary permeability of the fluorescein isothiocyanatedextran 40, a non-degraded permeation marker. When insulin solutions containing sulphobutyl ether-beta-cyclodextrin with a degree of substitution of the sulphobutyl group of 3.9 (SBE4-beta-CyD) were injected, the IRI level rapidly increased and maintained higher IRI levels for at least 8 h. The bioavailability of the insulin/SBE4-beta-CyD system was about twice that of insulin alone and approached 96%. The enhancing effects of SBE4-beta-CyD may be in part due to the inhibitory effects of SBE4-beta-CyDs on the enzymatic degradation and/or the adsorption of insulin onto the subcutaneous tissue at the injection site, although this does not apparently facilitate capillary permeability. These results suggest that SBE4-beta-CyD in aqueous insulin injection for subcutaneous administration is useful for improving the bioavailability and the hence the pharmacological effects of insulin.     

Thiolated carboxymethylcellulose: in vitro evaluation of its permeation enhancing effect on peptide drugs.

The purpose of this study was to evaluate the effect of sodium carboxymethylcellulose (NaCMC) and carboxymethylcellulose-cysteine (CMC-Cys) conjugates on the intestinal permeation of sodium fluorescein (NaFlu) and model peptide drugs, bacitracin and insulin. Cysteine was covalently linked to carbodiimide activated NaCMC. Iodometric titration of the polymer conjugates was used to determine the extent of immobilised cysteine. Permeation studies were performed on guinea pig small intestinal mucosa mounted in Ussing-type chamber. Unmodified NaCMC (1% m/v) significantly improved the transport ratio (R= P(app) polymer/ P(app) control) of NaFlu to 1.3 and 1% (m/v) NaCMC conjugated with cysteine further enhanced the permeation. Cysteine conjugation at 3.6, 5.3 and 7.3% (m/m) resulted in R-values of 1.4, 1.7 and 1.8, respectively. Decreasing the concentration of CMC-Cys, exhibiting 7.3% (m/m) of immobilised cysteine (CMC-Cys7.3) from 1% (m/v) to 0.5% (m/v) decreased the R-value of NaFlu from 1.8 to 1.2. NaCMC at 1% (m/v) in the presence of free cysteine had no significant effect on the R-value of NaFlu compared to NaCMC alone. Formulation of fluorescence labelled bacitracin and insulin in unconjugated NaCMC (1% m/v) did not significantly improve the permeation, however in the presence of 1% (m/v) CMC-Cys7.3 a significantly improved permeation was observed (R= 1.3). Conjugation at NaCMC with cysteine moieties significantly improves the intestinal permeation of the hydrophilic molecule NaFlu and the model peptide drugs bacitracin and insulin in vitro, therefore this conjugated system maybe useful for peroral administration of peptide drugs in the future.     

Effect of ethanol pretreatment on skin permeation of drugs

It has been demonstrated that ethanol (EtOH) can enhance skin permeation of drugs when simultaneously applied with drugs. However, only a few studies have reported on the pretreatment effect of EtOH on skin permeations. In this study, the pretreatment effects of EtOH on skin permeation of drugs were investigated by measuring changes in skin permeation and electrical skin resistance. Permeabilities of deuterium oxide (D2O), isosorbide mononitrate (ISMN), isosorbide dinitrate (ISDN), calcein sodium (CA-Na), and fluorescein isothiocyanate-dextran 4?kDa (FD-4, 3.3-4.4?kDa) were evaluated through Yucatan micropig skin pretreated with different concentrations of EtOH solution. From the results, almost constant skin permeabilities of D2O and ISDN were observed independent of EtOH concentration. Skin permeabilities of ISMN, CA, and FD-4 increased with low concentrations of EtOH, but decreased with high concentrations of EtOH. At 99.5% EtOH pretreatment, skin permeabilities of hydrophilic compounds (ISMN, CA, and FD-4) decreased to non-detectable levels. In addition, low molecular ion transports were almost constant at any EtOH concentration. Since molecular (ion) sizes of ISMN, CA, and FD-4 are larger than Na⁺, Cl^－, and D2O, permeation pathway sizes for hydrophilic compounds in the skin barrier may be remarkably decreased by pretreatment with high concentrations of EtOH. However, the permeability coefficient of ISDN was not influenced by any EtOH concentration, since ISDN is a lipophilic, low-molecular compound that permeated through the lipophilic stratum corneum pathway. The present results show useful information for repeatedly and topically applied formulations containing EtOH, and also contribute to the effective use of alcohol formulations.     

Age-related changes in skin permeability of hydrophilic and lipophilic compounds in rats

The effect of age on intact and stripped skin permeability of lipophilic (ketoprofen and isosorbide dinitrate) and hydrophilic permeants (deuterium oxide and diclofenac sodium) was investigated using STD: Wistar male rats aged 5 to 180 days. The permeability of permeants through intact skin increased with increasing lipophilicity of the permeants at each age, indicating that the permselective property of rat skin is revealed even at 5-days-old. The permeability coefficients through intact skin decreased with increasing age, and the extent of these decreases was higher for lipophilic permeants than that for hydrophilic permeants. On the other hand, the stripped skin permeability of permeants was almost the same at each age, and with aging each permeability coefficient through stripped skin decreased up to 21 days, dramatically during 21-90 days and then gradually again to 180 days. The thickness of the stratum corneum and stripped skin increased according to age with faster growth during 21-90 days. The reciprocal of the mean thickness of stratum corneum and stripped skin correlated well with intact skin and stripped skin permeability (r > 0.9), respectively. These results clarified that the permselectivity of rat skin against lipophilicity of permeant exists at the latest from 5 days after birth. In addition, it is speculated that the thickness of skin is a large factor in the decrease of its permeability with age.     

Dodecyl N,N-Dimethylamino Acetate and Azone Enhance Drug Penetration Across Human,Snake, and Rabbit Skin

The effectiveness of the penetration enhancers, dodecyl N, N-dimethylamino acetate (DDAA) and Azone, on pretreated human epidermis for the permeation of model drugs, indomethacin, 5-fluorouracil, and propranolol-HCl, was studied in in vitro diffusion cells. Snakeskin (Elaphe obsoleta) and rabbit pinna skin were compared as possible models for human skin. The drug concentrations were analyzed by HPLC. With all skins and all model drugs, DDAA increased drug permeability at least as well as Azone, and in most cases it was a more effective permeation enhancer. The relative permeation improvements in human skin, snakeskin, and rabbit skin were 10- to 20-, 5- to 50-, and 20- to 120-fold, respectively. Tritiated water served as an indicator of skin condition. Its penetration in the skin samples was independent of the drugs used, and both penetration enhancers significantly increased the flux of tritiated water through all skins. Thus, DDAA and Azone significantly increased the permeation of lipophilic and hydrophilic model compounds. Rabbit pinna skin was a poor model for human skin in vitro, while snakeskin was much closer to human skin in terms of transdermal permeability. In most cases drug permeability decreased in the order rabbit human > or < snake.     

应用A549细胞单层模型研究蛋白多肽类药物肺部吸收的特性应用A549细胞单层模型研究蛋白多肽类药物肺部吸收的特性

药物通过呼吸进行吸收的路径依次为气管→支气管→肺泡,其中通过肺泡上皮细胞吸收占90％以上。由于肺泡上皮细胞使大分子的蛋白多肽类药物不易通过,是它们渗透的主要屏障,因此对肺泡膜屏障特性、药物透过过程及渗透特性的研究有助于更     

Cyclodextrins and Drug Membrane Permeation: Thermodynamic Considerations

Cyclodextrins are hydrophilic oligosaccharides that can increase aqueous solubility of lipophilic drugs through formation of water-soluble drug/cyclodextrin complexes. Although the complexes are hydrophilic, and as such do not permeate biological membranes, the complexes are known to enhance drug permeation through lipophilic membranes and improve drug bioavailability after, for example, oral administration. However, it is not clear how cyclodextrins enhance the permeation. An artificial biomembrane (PermeaPad®) was used to study the effect of donor medium composition on drug permeation. It was observed that in aqueous solutions the hydrophilic cyclodextrins behave not like disperse systems but rather like organic cosolvents such as ethanol, increasing the solubility without having significant effect on the molecular mobility and ability of lipophilic drug molecules to partition into the lipophilic membrane. Also, that partition of dissolved drug molecules from the aqueous exterior into the membrane is at its maximum when their thermodynamic activity is at its maximum. In other words, that drug flux from aqueous cyclodextrin solutions through lipophilic membranes depends on both the concentration and the thermodynamic activity of dissolved drug. Maximum flux is obtained when both the drug concentration and thermodynamic activity of the dissolved drug molecules are at their maximum value.     

DEET-loaded solid lipid particles for skin delivery: in vitro release and skin permeation characteristics in different vehicles

DEET (N,N-diethyl m-toluamide) is a lipophilic compound which has a common use as an insect repellent and causes not only skin irritation but also systemic side effects at high concentrations in long-term skin application. In this study, DEET is incorporated into solid lipid particles, a colloidal drug delivery system, in order to reduce the percutaneous permeation and avoid toxic effects and also maintain drug effectiveness on the skin surface for a long duration of insect repellence. Solid lipid particles were prepared based on emulsion systems at different concentrations and after the characterization studies, the formulation with 20% lipid phase and 1:1 drug:lipid ratio was carried to in vitro release and skin permeation studies. Solid lipid particles with DEET were compared to free DEET using cream and hydrophilic gel vehicles. Results showed that incorporation of DEET into solid lipid particles reduced the release rate and skin permeation of DEET. Imaging studies using scanning electron microscopy showed that there were still solid lipid particles on skin surface after 2 h indicating that DEET could be present for a longer time on the application site.     

Improved paracellular uptake by the combination of different types of permeation enhancers

This study had the purpose to improve the paracellular uptake of drugs by combining the thiomer/reduced glutathione (GSH) permeation-enhancing system with a proteolytic enzyme. Due to the covalent binding of 2-iminothiolane to chitosan the thiomer chitosan-TBA (chitosan-4-thiobutylamidine) was obtained. Permeation studies were performed with freshly excised intestinal mucosa of guinea pigs mounted in Ussing-type chambers using on the one hand the low-molecular size marker flurescein (Na-Flu) and on the other hand the high-molecular size marker FITC-dextran. Apparent permeability coefficient (P(app)) as well as enhancement ratios (=P(app) permeation-enhancing system/P(app) control) were calculated. Trypsin, papain and bromelain displayed a permeation-enhancing effect for Na-Flu on the small intestinal mucosa. Enhancement ratios of 1.84, 1.63 and 1.78 were identified for 2% trypsin, 0.5% papain and 2% bromelain solutions, respectively. However, only bromelain could guarantee a significant permeation enhancement of FITC-dextran with a P(app) of 4.45+/-0.44 x 10(-6) cm/s representing an enhancement ratio of 1.57. A similar enhancement of FITC-dextran permeation was reached by the use of the chitosan-TBA (0.5%)/GSH (5%) system. Moreover, an additive permeation-enhancing effect of the chitosan-TBA/GSH system in combination with bromelain (2%) was observed, leading to a maximum P(app) of 5.91+/-0.51 x 10(-6) cm/s, which corresponds to an enhancement ratio of 2.1. According to these results, the combination of the thiomer/GSH system with bromelain might represent a new promising strategy in order to raise the in vivo efficacy of non-invasive administered hydrophilic macromolecular drugs.     

Improved α-Chymotrypsin Stability Upon Encapsulation in PLGA Microspheres by Solvent Replacement

PURPOSE: To investigate the potential of different solvents with better biocompatibility to replace CH2Cl2 in the encapsulation of alpha-chymotrypsin in poly (lactic-co-glycolic) acid (PLGA) microspheres without causing protein instability. METHODS: The oil-to-water (O:W) ratio in the emulsification step of the solid-in-oil-in-water (s/o/w) encapsulation process was optimized with respect to maximizing protein stability and encapsulation efficiency for various solvents. Formation of insoluble aggregates and residual enzyme activity were primarily used as stability parameters. Several solvents possessing low toxicity with different water solubility were used to prepare alpha-chymotrypsin loaded PLGA microspheres. RESULTS: The O:W ratio in the emulsification step is critical with respect to maintaining protein stability. This was related to the solvents' water solubility. In general, hydrophilic solvents were detrimental to protein stability and encapsulation efficiency. However, after optimization of the O:W ratio for solvents with different water solubility, protein stability was preserved during encapsulation using butyl acetate when poly (ethylene glycol) (PEG) was used as the emulsifying agent (ca. 1% of non-covalent aggregates and 93 +/- 10% of residual specific activity). CONCLUSIONS: The s/o/w technique was successfully improved by replacing the ICH class 2 solvent CH2Cl2 with the class 3 solvent butyl acetate without compromising alpha-chymotrypsin stability.     

Development of an in vitro blood-brain barrier model based on immortalized porcine brain microvascular endothelial cells

Immortalized porcine brain microvessel endothelial cells (PBMEC/C1-2) were used to develop a model for measurement of blood-brain barrier permeation of central nervous system active drugs. Previous studies showed that a system using C6 astrocyte glioma conditioned medium leads to cell layers with transendothelial electrical resistance values up to 300 Omega cm(2) and a permeability coefficient P(e) of 3.24 +/- 0.14 x 10(-4) cm/min for U-[(14)C]sucrose, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro. However, commercially available inserts for the Transwell system were not permeable for highly lipophilic compounds, such as diazepam. Systematic studies with different insert showed, that inserts with a pore width of 1 microm proved to be optimal for permeation studies of lipophilic compounds. Permeability studies with a set of three benzodiazepines further supported this finding.