Novel Bioadhesive Chitosan-EDTA Conjugate Protects Leucine Enkephalin from Degradation by Aminopeptidase N

Purpose. To develop a novel bioadhesive polymer that protects peptide drugs from luminal degradation by aminopeptidase N and to evaluate the system in vitro on porcine mucosa. Methods. EDTA was covalently bound to chitosan in order to combine the bioadhesive properties of the polymer with the well known capacity of EDTA to complexe metal ions which are essential for the enzymatic activity of proteases. The inhibitory effect of this polymer conjugate was evaluated by using leucine enkephalin (Leu enkephalin) as a model drug. The degree of Leu enkephalin degradation caused by aminopeptidase N (EC 3.4.11.2), as well as porcine mucosa, in the presence of the polymer conjugate, was quantified by HPLC analysis. Results. The chitosan-EDTA conjugate is capable of binding 2.01 ± 0.12 mmole of zinc per gram of polymer at pH 6.5 (n = 3; ± S.D.). As zinc is an essential co-factor for aminopeptidase N, enzyme activity (48 mU/ml) could be completely inhibited under the use of 1.0% chitosan-EDTA conjugate. The inhibitory effect of 1.0% chitosan-EDTA conjugate on the degradation of Leu enkephalin on porcine mucosa within 3 h at 37°C was even 2.9-fold higher than that of a recently developed zinc complexing bacitracin-poly(acrylic acid) conjugate of the same concentration. The novel polymer conjugate is more bioadhesive than unmodified chitosan and is easily hydratable in water and basic aqueous solutions exhibiting quick swelling properties. Conclusions. The bioadhesive polymer conjugate described here seems to be a useful tool in overcoming enzymatic degradation by aminopeptidase N.     

Drug Release from pH and Ionic Strength Responsive Poly(acrylic acid) Grafted Poly(vinylidenefluoride) Membrane Bags In Vitro

Pharmaceutical Research -     

pH-and thermo-sensitive pluronic/poly(acrylic acid) in situ hydrogels for sustained release of an anticancer drug

In this study, we developed oral in situ gelling formulations composed of pluronic (Plu) and polyacrylic acid (PAA) for the delivery of an anticancer drug, epirubicin (Epi). We investigated various Plu/PAA/Epi formulations for their physicochemical properties and in vitro permeation and accumulation, as well as for in vivo pharmacokinetic and antitumor efficacy. A scanning electron microscopic (SEM) image of Plu 14%/PAA 0.75%/Epi hydrogel showed a sponge-like structure. This formulation has suitable gelation time, water content, bioadhesive force, structural stability, and a high permeation percentage of Epi, with sustained drug release characteristics for 96?h. This hydrogel was retained at the end of the ileum near the colon of Sprague-Dawley (SD) rats for at least 12?h. An in vivo pharmacokinetic study using SD rats showed that after oral administration in this formulation, Epi had prolonged half-life, greater area under the curve, and higher relative bioavailability than in an oral Epi solution. In vivo tumor growth inhibition of Epi in this formulation was more pronounced compared with oral Epi and intravenous Epi solutions in CT-26 mouse colon adenocarcinoma bearing Balb/c mice. This study highlights the advantages of using oral in situ temperature- and pH-sensitive hydrogels for future cancer therapy.     

Evaluation of Mucoadhesive Polymers in Ocular Drug Delivery. II. Polymer-Coated Vesicles

Association of Carbopol 934P and Carbopol 1342 (a hydrophobic modified Carbopol resin) with phospholipid vesicles was assessed by photon correlation spectroscopy and microelectrophoresis at pH 7.4 and 5. The precorneal clearance of the polymer-coated vesicles was compared to that of uncoated vesicles by lacrimal dacryoscintigraphy in the rabbit. The mucoadhesive polymer-coated vesicles demonstrated significantly enhanced precorneal retention compared to noncoated vesicles only at pH 5 (P < 0.005). The entrapment and subsequent release of tropicamide from Carbopol 1342-coated and uncoated liposomes were determined in vitro together with an in vivo evaluation of the vesicles formulated at the lower pH. Mucoadhesive polymer-coated vesicles failed to increase significantly the bioavailability of the entrapped tropicamide compared to uncoated vesicles and aqueous solution.     

Evaluation of Mucoadhesive Polymers in Ocular Drug Delivery. I. Viscous Solutions

The potential of a mucoadhesive polymer as an ophthalmic vehicle is evaluated within the rabbit. Precorneal clearance of a mucoadhesive polymer solution (Carbopol 934P) is compared to that of an equiviscous nonmucoadhesive poly(vinyl alcohol) solution (PVA) and buffer (PBS). The precorneal retention of the Carbopol 934P, as studied by lacrimal dacryoscintigraphy, is shown to be significantly greater (P < 0.05) than that of PVA, which, in turn, is significantly greater than that of PBS. The effect of the polymer solution on the bioavailability of pilocarpine is subsequently assessed by measuring the relative miotic response intensities produced by a 1% solution of the drug. Carbopol 934P solution produces a significant increase (P < 0.05) in bioavailability as compared to PVA and PBS. The bioavailability from PVA is significantly greater (P < 0.05) than that from PBS. Studies evaluating vehicle-drug association indicated no binding of the drug to the polymer.     

A Novel in Vitro Delivery System for Assessing the Biological Integrity of Protein upon Release from PLGA Microspheres

Purpose. The development of a novel in vitro system is required to assess the stability and release kinetics of a protein microsphere formulation used for drug delivery to the brain. Methods. Microspheres containing lysozyme as model protein were prepared using a (w/o/w) emulsion-solvent evaporation process. Both the active and total (active + inactive) encapsulation efficiencies and release profiles were determined. The biologic activity of lysozyme was measured using bacterial cell lysis; total protein content was measured using a ¹²⁵I-radiolabel. A novel in vitro apparatus was developed to determine kinetics over a sustained time period (>30 days). Results. The microencapsulation technique allowed an entrapment of active lysozyme at 80 ± 4% and a sustained (>42 days) in vitro release. The kinetics study showed that the novel in vitro system was able to detect the release of low amounts (ng) of protein. To improve the stability of the protein within microspheres and allow the release of biologically active lysozyme, a basic additive ( Mg(OH)₂ ) was successfully encapsulated. Conclusions. This novel in vitro system was appropriate to study protein microsphere release kinetics. In addition, the model is cost-effective and mimes brain physiological conditions more closely than previous models.     

Mucoadhesive Polymers in Peroral Peptide Drug Delivery. II. Carbomer and Polycarbophil Are Potent Inhibitors of the Intestinal Proteolytic Enzyme Trypsin

Purpose. The evaluation of the inhibitory action of two mucoadhesive poly(acrylates), polycarbophil and carbomer, registered by the Food and Drug Administration (FDA), on the intestinal proteolytic enzyme trypsin. Methods. The effect of the polymers on trypsin activity by measuring the degradation of a trypsin specific substrate. Binding of Ca²⁺ ions and proteins (¹²⁵I-BSA) to the poly(acrylates). The influence of the polymers on the secondary trypsin structure by circular dichroism. Results. Trypsin inhibition was found to be time-dependent upon addition of Ca²⁺ in the degradation experiment. Only when Ca²⁺ was added within 10 min after trypsin incubation, recovery of the enzyme could be observed. Both polymers showed a strong Ca²⁺ binding ability. Carbomer, which had a higher inhibitory effect on trypsin activity, also revealed a higher Ca²⁺ binding affinity than polycarbophil. The amount of Ca²⁺ depleted out of the trypsin structure and the reduction of enzyme activity were comparable. Immobilization of trypsin by binding to the polymers could not be observed at pH 6.7. Circular dichroism studies suggested that, under depletion of Ca²⁺ from trypsin, the secondary structure changed its conformation, followed by an increased autodegradation of the enzyme. Conclusions. The poly(acrylates) investigated may have potential to protect peptides from tryptic degradation and may be used to master the peroral delivery of peptide drugs.     

Chemical Interactions Between Drugs Containing Reactive Amines with Hydrolyzable Insoluble Biopolymers in Aqueous Solutions

Chemical reactions between drugs containing reactive amines with hydrolyzable polymers in buffer solutions were investigated. Phenylalkylamines with increasing nucleophilic reactivity were used as model drugs. Solutions of phenylalkylamines were reacted heterogeneously with representative biodegradable polyanhydride and polyester powders in various pH solutions, and the recovery of the amines from the solutions was determined. Poly(sebacic acid), a reactive polyanhydride, reacted by amide formation with the tested amines and their respective HC1 salts when exposed to physiologic pH (pH 7.4). However, at pH 5.0 no interaction occurred. The aromatic polyanhydride, PCPP, and the polyesters based on lactic acid and caprolactone did react with the amine derivatives at pH 7.4, but at a slower rate. The reaction can be avoided with appropriate salt derivatives of the amines.     

Preparation and evaluation of thiomer nanoparticles via high pressure homogenization

The aim of this study was to establish and evaluate a high pressure homogenization method for the preparation of thiomer nanoparticles. Particles were formulated by incorporation of the model protein horseradish peroxidase in chitosan-glutathione (Ch-GSH) and poly(acrylic acid)-glutathione (PAA-GSH) via co-precipitation followed by air jet milling. The resulting microparticles were suspended in distilled water using an Ultraturax and subsequently micronized by high pressure homogenization. Finally, resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity and release behaviour. The mean particle size after 30 cycles with a pressure of 1500 bar was 538 ± 94 nm for particles consisting of Ch-GSH and 638 ± 94 nm for particles consisting of PAA-GSH. Nanoparticles of Ch-GSH had a positive zeta-potential of +1.03 mv, whereas nanoparticles from PAA-GSH had a negative zeta potential of ?6.21 mv. The maximum protein load for nanoparticles based on Ch-GSH and based on PAA-GSH was 45 ± 2% and 37 ± %, respectively. The release profile of nanoparticles followed a first order release kinetic. Thiolated nanoparticles prepared by a high pressure homogenization technique were shown to be stable and provide controlled drug release characteristics. The preparation method described here might be a useful tool for a more upscaled production of nanoparticulate drug delivery systems.     

Swelling and Dissolution Kinetics During Peptide Release from Erodible Anionic Gel Beads

The transient dynamic swelling and dissolution behavior during the release of a growth hormone releasing peptide, [D-Trp²-D-Phe⁵]GHRP, from credible, non-cross-linked poly(methyl methacry-late-co-methacrylic acid) (PMMA/MAA) beads has been investigated at pH 7.4 as a function of buffer concentration. Although the swelling front penetration shows a ionization-limited behavior similar to that of nonerodible cross-linked PMMA/MAA beads, the normalized diameter of the polymer beads exhibits a brief initial rise followed by an extended linear decline due to establishment of the polymer dissolution process. This is consistent with the general kinetic scheme of dissolution of glassy polymers originally predicted for the slab geometry. In all cases, the initial gel thickness increases as a result of the ionization and swelling of the glassy PMMA/MAA beads. This is followed by an extended period of constant gel thickness due to the onset of polymer dissolution and the synchronization of movement of the swelling and dissolution fronts. The resulting constant gel layer thickness as well as the onset and duration of front synchronization shows an increasing trend with decreasing buffer concentrations. As a result, the corresponding peptide release is slower and the release duration longer at lower buffer concentrations. This is believed to be the first time that a synchronization of swelling and dissolution fronts has been documented for a spherical credible sample. Although such synchronization of fronts does not result in a constant rate of peptide release due to the spherical geometry, some non-Fickian release characteristics have been observed.     

In Vivo Properties of an In Situ Forming Gel for Parenteral Delivery of Macromolecular Drugs

Purpose. This study characterizes the in vivoproperties of an in situforming gel, comprising an IPC of water-soluble polymers, PMA and PEG, for sustained release of macromolecular drugs. Methods. 40, 50 or 60% w/v formulations were injected subcutaneously in a rat model either alone, or containing model macromolecules, 3A2-ATG-psODN or REV-psODN, to (i) determine the approximate gelling and residence time of the gel at the site of injection, (ii) assess the biological efficacy of the formulation using a MZ sleep time model, and (iii) demonstrate specificity of the sequence and selectivity of the psODNs by measuring changes in microsomal enzyme levels and urine volumes. Results. A sol to gel transition requires 15 min in vivo, and the 60% w/v IPC gel remains at the site of injection for up to 72 hr. The MZ sleep times and CYP3A2 expression due to 3A2-ATG-psODNs released from the gel are significantly different compared to that of REV-psODNs. Conclusions. The IPC solutions exhibit phase transformation in vivo, and demonstrate no evidence of toxicity. The pharmacological effects observed from the of release of 3A2-ATG-psODNs suggest that the formulation can entrap, protect, and sustain the delivery of macromolecules.     

谈国内合成新药的研究与开发

目的 :为研究开发国产合成新药提供参考建议。方法 :总结研究开发10个合成新药的实践经验。结果与结论 :产学研结合开发新药促进了科研成果的产业化     

Mechanisms of Mucoadhesion of Poly(acrylic Acid) Hydrogels

It has been proposed that mucoadhesives which adhere to the gastric mucus layer may be used to prolong gastric retention time of oral dosage forms. Preliminary studies, using acrylic hydrogels, have established that the density of carboxyl groups on the polymer chain is important for mucoadhesion. To understand the role(s) of the carboxyl groups in mucoadhesion, acrylic acid–aerylamide random copolymers [P(AA-co-AM)] were synthesized, and the adhesion strength of the cross-linked polymers to the gastric mucus layer was examined as a function of the pH, initial concentration of the cross-linking agent, degree of swelling, and carboxyl-group density. From the study on mucoadhesion of various P(AA-co-AM), it was found that at least 80% of the vinyl groups of the polymer must possess carboxyl groups in the protonated form. The dependence of mucoadhesion on pH and carboxyl-group density suggests that mucoadhesion occurs through hydrogen bonding. In addition, the density of the cross-linking agent significantly affects mucoadhesion. As the density of the cross-linking agent is lowered, the mucoadhesive strength increases, although the density of carboxyl groups in the test surface area is reduced. It is concluded that for mucoadhesion to occur, polymers must have functional groups that are able to form hydrogen bonds above the critical concentration (80% for vinyl polymers), and the polymer chains should be flexible enough to form as many hydrogen bonds as possible.     

Behavioral Development in Children Prenatally Exposed to Drugs and Alcohol

Empirical research on the behavioral consequences to the offspring of use of recreational and addictive drugs and alcohol by pregnant women is reviewed. The current epidemic of cocaine use has raised the specter of a host of “cocaine babies” whose prenatally induced impairments will interfere with social and academic functioning and constitute an immense social burden. In fact, examination of effects of drug exposure on infant behavior and subsequent development suggests a much more subtle and complicated process which must take into account not only the child's prenatal exposure but the various other environmental factors which contribute to eventual outcome. These other factors include caregiving competence and social environment. [Translations are provided in the International Abstracts Section of this issue.]     

In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates

Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid) (PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation-enhancing effect of unmodified PAA and PAA₂₅₀-Cys_, displaying 580 μmol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA₂₅₀ and 0.5% (w/v) PAA₂₅₀-Cys was 1.3- and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4% (w/v) unmodified PAA₂₅₀ or 4% (w/v) thiolated PAA₂₅₀ were orally given to rats. The PAA₂₅₀-Cys solution increased the area under the plasma concentration-time curve (AUC_0-12) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA₂₅₀. This in vivo study revealed that PAA₂₅₀-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.     

A Comparison of the Physical Stability of Amorphous Felodipine and Nifedipine Systems

Purpose The objective of this study was to investigate thermodynamic and kinetic factors contributing to differences in the isothermal nucleation rates of two structurally related calcium channel blockers, nifedipine and felodipine, both alone and in the presence of poly(vinylpyrrolidone) (PVP).Materials and Methods Thin films of amorphous systems were cast onto glass slides and the nucleation rate was determined using optical microscopy. Enthalpy, entropy, and free energy of crystallization of the pure compounds were measured using differential scanning calorimetery (DSC). Molecular mobility and glass transition temperature of each amorphous system were characterized using DSC and hydrogen bonding patterns were analyzed with infrared spectroscopy. The composition dependence of the thermodynamic activity of the amorphous drug in the presence of the polymer was estimated using Flory‐Huggins lattice theory.Results Nifedipine crystallized more readily than felodipine from the metastable amorphous form both alone and in the presence of PVP despite having a similar glass transition temperature and molecular mobility. Nifedipine was found to have a larger enthalpic driving force for crystallization and a lower activation energy for nucleation.Conclusions The properties of the metastable form alone did not explain the greater propensity for nifedipine crystallization. When considering the physical stability of amorphous systems, it is important to also consider the properties of the crystalline counterpart.     

蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球研究进展

缓释微粒给药系统是蛋白质/多肽药物传输系统的一个重要研究方向，聚乳酸和乳酸-羟基乙酸共聚物是制备缓释微球最常用的载体材料。蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球常用的制备方法包括溶剂萃取/挥发法(复乳法)、相分离法和喷雾干燥法。本文总结了微球制备中面临的难点如蛋白质/多肽药物稳定性、包封率、药物突释和药物吸附等问题，并综述了保持药物结构稳定性和生物活性、提高包封率、改善药物释放曲线等微球制备方法和进展。     

Visualization of Insulin-Loaded Nanocapsules: In Vitro and in Vivo Studies After Oral Administration to Rats

Purpose. Biodegradable poly(isobutylcyanoacrylate) nanocapsules have been recognized as a promising carrier for oral administration of peptides and proteins. In the present study, we investigate the fate of insulin-loaded nanocapsules by fluorescence and transmission electron microscopy (TEM) after intragastric force-feeding to rats. Methods. Insulin-, Texas-red^®-labeled insulin, or gold-labeled insulin-loaded nanocapsules were first characterized. Rats received a single dose of nanocapsules (diameter 60-300 nm, 57 IU insulin/kg) by intragastric force-feeding. After 90 min, ileum was isolated and prepared for fluorescence and transmission electron microscopy. Results. Nanocapsules were observed on both sides of the gut epithelium and in blood capillaries. In M-cell-free epithelium, apparently intact nanocapsules could be seen in the underlying tissue, suggesting they could cross the epithelium and carry the encapsulated peptide. In M-cell-containing epithelium, nanocapsules appeared degraded in the vicinity of macrophages. It is noteworthy that intestinal absorption of nanocapsules was observed without artifacts forcing the nanocapsules to stay in the gut. Conclusions. Based on TEM observations, this study shows the intestinal absorption of biodegradable nanocapsules leading to the transport of insulin across the epithelium mucosa. The fate of the nanocapsules appeared different depending on the presence or the absence of M cells in the intestinal epithelium.     

Curcumin-Loaded PLA Nanoparticles: Formulation and Physical Evaluation

Curcumin is a polyphenolic compound derived from Curcuma domestica (Zingiberaceae) that possesses diverse pharmacological effects including anti-inflammatory, antioxidant, antimicrobial, and anticarcinogenic activities. Although phase I clinical trials have shown curcumin as a safe drug even at high doses (12 g/day) in humans, poor bioavaibility largely limits its pharmacological activity. Nanoencapsulation in biodegradable polymers is a promising alternative to improve curcumin bioavaibility. In this study, curcumin was encapsulated in biodegradable polymer poly-(lactic acid) (PLA) nanoparticles via the emulsification-solvent evaporation method. Optimization of selected parameters of this method including the type of solvent, surfactant concentration, drug loading, sonication time, and centrifugation speed, were performed to obtain polymeric nano-carriers with optimum characteristics. Dichloromethane was used as the solvent and vitamin E polyethylene glycol succinate (TPGS) was used as the surfactant. Four minutes of sonication time and centrifugation at 10500 rpm were able to produce spherical nanoparticles with average size below 300 nm. The highest encapsulation efficiency was found on PLA nanoparticles containing 5% of curcumin at 89.42 ± 1.04%. The particle size, polydispersity index, zeta potential of 5% curcumin-PLA nanoparticles were 387.50 ± 58.60 nm, 0.289 ± 0.047, and −1.12 mV, respectively. Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD) studies showed partial interaction between the drug and polymer.     

Poly(2-Ethyl-2-Oxazoline) as an Alternative to Poly(Vinylpyrrolidone) in Solid Dispersions for Solubility and Dissolution Rate Enhancement of Drugs

Poly(2-ethyl-2-oxazoline) (PEOX), a biocompatible polymer considered as pseudopolypeptide, was introduced as a potential alternative to the commonly used polymer, poly(vinylpyrrolidone) (PVP) for the preparation of solid dispersion with a poorly soluble drug. Glipizide (GPZ), a Biopharmaceutical Classification System class II model drug, was selected for solubility and dissolution rate study. GPZ-polymer solid dispersions and physical mixtures were characterized and investigated by X-ray diffractometry, differential scanning calorimetry, scanning electron microscopy, and FTIR spectroscopy. The impact of polymers on crystal nucleation kinetics was studied, and PEOX exhibited strong inhibitory effect compared with PVP. Solubility and dissolution behavior of the prepared solid dispersions and their physical blends were in vitro examined and evaluated. A significant enhancement in GPZ solubility was obtained with PEOX compared with the pure drug and solid dispersion with PVP. A big improvement in the intrinsic dissolution rate (45 times) and dissolved amount of GPZ (58 times) was achieved with PEOX in fasted state simulated intestinal fluid, against comparable enhancement observed with PEOX and PVP in phosphate buffer at pH 6.8. Lower molecular weight of PEOX-5K (5000 g/mol) was found to be superior to higher molecular weight PEOX-50K (50,000 g/mol) in the improvement of dissolution behavior. The findings of this study with GPZ as a model drug introduce lower molecular weight PEOX as a promising polymeric carrier toward better oral bioavailability of poorly soluble drugs.