Formulations for protecting the probiotic Saccharomyces boulardii from degradation in acidic condition

Saccharomyces boulardii is a nonpathogenic yeast with proven health benefits, some of them depending on its viability. However, the living yeast is sensitive to environmental conditions and its viability is less than 1% in the faeces after oral administration. Therefore, we assessed the survival conditions of S. boulardii in aqueous suspension and in its freeze-dried form and we formulated microspheres with the former and tablets with the latter in order to preserve the viability of the probiotic. While the viability of the yeast in aqueous suspension could be maintained for one year at -20 degrees C and +5 degrees C, increasing the temperature led to almost total mortality within 14 d at +40 degrees C and 4 d at +60 degrees C. The viability of the freeze-dried yeast was preserved for one year at +25 degrees C without moisture. With 75% relative humidity, the mortality was significant at 28 d at +25 degrees C and almost total within 1 d at +60 degrees C. In vitro, whereas less than 1% of non-encapsulated or non-tabletted S. boulardii survived after 120 min at pH 1.1, both formulations in microspheres and direct compression enabled to protect the yeast from degradation in HCl and to release it viable at pH 6.8. However, despite a similar release profile from both dosage forms, the compression led to a significant decrease in the viability of the freeze-dried yeast. In conclusion, although both formulations are efficient in protecting S. boulardii in acidic condition, microspheres provide a higher entrapment efficiency and a faster release of the viable probiotic in intestinal condition than matrix tablets.     

Site-Specific Drug Delivery to Pilosebaceous Structures Using Polymeric Microspheres

In order to improve the therapeutic index of adapalene, a new drug under development for the treatment of acne, site-specific delivery to the hair follicles using 50:50 poly(DL-lactic-co-glycolic acid) microspheres as particulate carriers was investigated in vitro and in vivo. The percutaneous penetration pathway of the microspheres was shown to be dependent on their mean diameter. Thus, after topical application onto hairless rat or human skin, adapalene-loaded microspheres (5-µm diameter) were specifically targeted to the follicular ducts and did not penetrate via the stratum corneum. The in vitro release of adapalene from the microspheres into artificial sebum at 37°C was controlled and faster than the in vivo sebum excretion in humans. Aiming to reduce either the applied dose of drug or the frequency of administration, different formulations of adapalene-loaded microspheres were evaluated in vivo in the rhino mouse model. A dose-related comedolytic activity of topical formulations of adapalene-loaded microspheres was observed in this model. Furthermore, by applying a site-specific drug delivery system (0.1% adapalene) every other day or by administering a 10-fold less concentrated targeted formulation (0.01%) every day, a pharmacological activity equivalent to a daily application of an aqueous gel containing drug crystals (0.1% adapalene) was observed. Since an aqueous gel containing 10% adapalene-loaded microspheres was not irritating in a rabbit skin irritancy test, this formulation was applied onto forearms of human volunteers. Site-specific drug delivery was further evidenced by follicular biopsy. These results support the view that follicular drug targeting using 5-µm polymeric microspheres may represent a promising therapeutic approach for the treatment of pathologies associated with pilosebaceous units.     

Morphine-induced Neuroimmunomodulation in Murine Visceral Leishmaniasis: The Role(s) of Cytokines and Nitric Oxide

Opioid modulation of host resistance to infectious diseases is well documented; however, not much is known during visceral leishmaniasis (VL). Low doses of morphine, administered subcutaneously in Leishmania donovani-infected BALB/c mice, on days 0 and +15, significantly (p < 0.05) suppressed (1 mg/kg/day) or even sterile-cleared (2 mg/kg/day) the infection; paradoxically, high doses (10 and 30 mg/kg/day) exacerbated the infection. In vitro, low concentration (1 × 10⁻⁹ and 1 × 10⁻¹¹ M) morphine treatment of L. donovani-infected mouse peritoneal macrophages (PM), endowed them with significant (p < 0.05) leishmanicidal activity, whereas a high-concentration (1 × 10⁻⁵ M) treatment augmented intramacrophage parasite growth. Naloxone pre-treatment of infected-mice (4 mg/kg × 2) and of infected-PM (1 × 10⁻⁵ M), blocked only the morphine low dose/concentration-induced protective effect. The splenocytes from protected mice and morphine low concentration-treated infected-PM, elaborated significantly (p < 0.05) enhanced levels of interleukin-12, interferon-γ, tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor and nitrite in the culture medium; a high dose/concentration suppressed their elaboration. Curiously, only morphine high dose/concentration-treated infected mice splenocytes and infected PM, produced significantly (p < 0.05) increased quantity of transforming growth factor-β1. Aminoguanidine, significantly (p < 0.05) blocked the morphine low dose/concentration-induced protective effect, in vivo and in vitro. This first study demonstrates dose-dependent biphasic modulatory effects of morphine in L. donovani-infected mice and PM, in vitro, apparently via nitric oxide-dependent mechanisms. These results thus demonstrate the implications of opiate abuse on the efficacy assessment of antileishmanial drugs and vaccines, and on the reactivation of latent VL in areas where both drug abuse and VL are rampant. Disclaimers: nil Sources: Senior Research Fellowship to P. S. National Eligibility Test, Senior Research Fellow, The Council of Scientific and Industrial Research, New Delhi, India. Drugs. morphine sulphate (Government Opium and Alkaloid Factory, Ghazipur, India). Meeting presentation. part of the work presented in 12th Society on NeuroImmune Pharmacology Conference, Santa Fe, New Mexico, USA, April 05–9, 2006.     

Design and characterization of chitosan-alginate microspheres for ocular delivery of azelastine

Abstract

The use of mucoadhesive biopolymers is one of the best approaches to prolong the drug residence inside the cul-de-sac, consequently increasing the bioavailability. Thus, the focus of this work was to develop mucoadhesive microspheres to overcome the limitations of ocular drug delivery. The chitosan-sodium alginate microspheres of azelastine hydrochloride were fabricated using modified ionotropic gelation technique. The particle size, zeta potential, entrapment efficiency and drug release kinetics were evaluated and characterized by SEM, FT-IR, DSC, in vitro mucoadhesion and in vivo study. The microspheres had average particle size in the range of 3.55 to 6.70?µm and zeta potential +24.55 to +49.56?mV. The fabricated microspheres possess maximum drug entrapment of 73.05% with 65% mucin binding efficiency and revealed a controlled release over the 8-h period following a non-Fickian diffusion. SEM showed that microspheres were distinct solid with irregular shape. FT-IR and DSC results concluded the drug entrapment into microspheres. In vivo studies on ocular rat model revealed that azelastine microspheres had better efficacy. Chitosan sodium alginate microspheres prepared were in particle size range suitable for ocular purpose. In vitro release and in vivo efficacy studies revealed that the microspheres were effective in prolonging the drug’s presence in cul de sac with improved therapeutic efficacy.     

Pharmacological evaluation of C-3 modified Betulinic acid derivatives with potent anticancer activity

Summary In vitro and in vivo pharmacological screening of Betulinic acid (BA) and five dihydro-BA derivatives modified at C-3 position [4-nitrobenzyl-oximino (1), 2-4-difluoro-benzoyloxy (2), 2-4-difluoro-benzylidene-amino (3), benzoyl-hydrazono (4), and 4-fluorophenyl-hydrazono (5)], having potent in vitro anti-cancer activity was carried out using ADME, animal PK and tumor studies. We found that BA and the derivatives had poor aqueous solubility (<0.1 μg/ml), low to moderate permeability (log Pe < −5.0) and high plasma protein binding (>70%). Although BA and 5 were metabolized by human liver microsomes, derivatives 1, 2, 3 and 4 possessed good in vitro metabolic stability. Except 3 which inhibited CYP1A2 isoform by more than 50% none of the other compounds inhibited key cytochrome P450 enzyme isoforms (CYP1A2, CYP2C9, CYP2D6 and CYP3A4) at 10 μM. Based on in vitro results one derivative 1 was tested in rodent PK and tumor studies. We found that 1 exhibited favorable pharmacokinetic characteristics of a systemically administered drug and showed better in vivo anti-tumor efficacy as compared to BA in a human colon cancer xenograft model. Our results show that BA derivatives are potential anti-cancer compounds which need to be explored in detail.     

Preclinical and Clinical <Emphasis Type="BoldItalic">In Vitro In Vivo</Emphasis> Correlation of an hGH Dextran Microsphere Formulation

Purpose To investigate the in vitro in vivo correlation of a sustained release formulation for human growth hormone (hGH) based on hydroxyethyl methacrylated dextran (dex-HEMA) microspheres in Pit-1 deficient Snell dwarf mice and in healthy human volunteers. Materials and Methods A hGH-loaded microsphere formulation was developed and tested in Snell dwarf mice (pharmacodynamic study) and in healthy human volunteers (pharmacokinetic study). Results Single subcutaneous administration of the microspheres in mice resulted in a good correlation between hGH released in vitro and in vivo effects for the hGH-loaded microsphere formulation similar to daily injected hGH indicating a retained bioactivity. Testing the microspheres in healthy volunteers showed an increase (over 7–8 days) in hGH serum concentrations (peak concentrations: 1–2.5 ng/ml). A good in vitro in vivo correlation was obtained between the measured and calculated (from in vitro release data) hGH serum concentrations. Moreover, an increased serum concentration of biomarkers (insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3) was found again indicating that bioactive hGH was released from the microspheres. Conclusions Good in vitro in vivo correlations were obtained for hGH-loaded dex-HEMA microspheres, which is an important advantage in predicting the effect of the controlled drug delivery product in a clinical situations.     

The Effects of Hexetidine (Oraldene™) on the Adherence of Candida Albicans to Human Buccal Epithelial Cells In Vitro and Ex Vivo and on In Vitro Morphogenesis

Purpose. This study reports the effects of hexetidine (Oraldene) on two virulence attributes of Candida albicans, namely,in vitro and ex vivoadherence of yeast cells to buccal epithelial cells (EEC) and in vitro morphogenesis. Methods. The effects of hexetidine treatment of either yeast cells (stationary and exponential phases) or BEC on Candidal adherence, in terms of viable and non-viable adherent yeast cells, were evaluated using an acridine orange stain in conjunction with fluorescence microscopy. Ex vivoanti-adherence effects were determined by rinsing BEC in vivo with hexetidine (0.1%), removal of BEC after defined periods and inclusion in the adherence assay. The effects of hexetidine on morphogenesis were evaluated using light microscopy. Yeast cell viability following exposure to a range of concentration of hexetidine (0.005-0.1 % v/v) for defined periods was determined following serial dilution and enumeration on solid media. Results. Treatment of stationary and exponential phase yeast cells or BEC with hexetidine (0.1%) for a range of times (10–300 s) or, alternatively, with a range of concentrations of hexetidine (0.005–0.1 %) for a fixed time (30s) significantly decreased the resultant Candidal/ epithelial adhesion. No correlations were observed between reduced adherence and either time of treatment or hexetidine concentration. In vivotreatment of BEC with hexetidine (0.1%) for 30s resulted in prolonged and significant reductions in the ex vivo adherence of both viable and non-viable yeast cells for periods of up to (and including) four hours post-rinsing. Treatment of C. albicans blastospores with hexetidine (0.05, 0.1% v/v) for 10s and 30s totally inhibited Candida morphogenesis, whereas treatment with lower antiseptic concentrations significantly reduced the extent of Candida morphogenesis and the rate of hyphal development. The effects of hexetidine on yeast cell viability were both concentration and time-dependent. Conclusions. The reduced adherence of C. albicans to BEC and the modification or inhibition of morphogenesis following exposure to hexetidine suggests a clinical role for hexetidine in the prophylaxis of both superficial candidosis and the systemic complications resulting from invasion of sub-epithelial tissue.     

Targeted delivery of diclofenac sodium via gelatin magnetic microspheres formulated for intra-arterial administration

In the present work, we have attempted to deliver diclofenac sodium to a target site by intra-arterial injection of gelatin magnetic microspheres and subsequent localization using an external magnet. Drug-loaded magnetic microspheres were prepared by emulsification/cross-linking method, characterized by drug loading, magnetite content, size distribution, optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) analysis, differential scanning calorimetry (DSC), X-ray diffraction (XRD), absence of glutaraldehyde by gas chromatography, and in vitro release studies. The targeting efficiency and the therapeutic efficacy of microspheres were studied in vivo in rabbits. The microspheres showed drug loading of 9.1, 18.7, 24.9% w/w, magnetite content of 27.8–28.9% w/w with an average size range of 25–30.6 μm, depending upon the drug–polymer ratio. They were spherical in nature as evidenced by optical microscopy and SEM. FT-IR, DSC, and XRD studies revealed the absence of drug–polymer interaction. Gas chromatography confirmed the absence of residual glutaraldehyde. The microspheres were able to prolong the drug release over 24–30 days and the application of sonication during in vitro release study has slightly increased the release rate. After intra-arterial administration of microspheres, 77.7% of injected dose was recovered at the target site which revealed good targeting efficiency. The microspheres effectively reduced joint swelling, but lesser extent than the oral diclofenac sodium in high dose, in antigen induced arthritic rabbits without producing gastric ulceration which was observed in rabbits treated with oral diclofenac sodium.     

Coated whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast

Viable Saccharomyces boulardii, used as a biotherapeutic agent, was encapsulated in food-grade whey protein isolate (WP) and alginate (ALG) microparticles, in order to protect and vehicle them in gastrointestinal environment. Yeast-loaded microparticles with a WP/ALG ratio of 62/38 were produced with high encapsulation efficiency (95%) using an extrusion/cold gelation method and coated with ALG or WP by a simple immersion method. Swelling, yeast survival, WP loss and yeast release in simulated gastric and intestinal fluids (SGF and SIF, pH 1.2 and 7.5) with and without their respective digestive enzymes (pepsin and pancreatin) were investigated. In SGF, ALG network shrinkage limited enzyme diffusion into the WP/ALG matrix. Coated and uncoated WP/ALG microparticles were resistant in SGF even with pepsin. Survival of yeast cells in microparticles was 40% compared to 10% for free yeast cells and was improved to 60% by coating. In SIF, yeast cell release followed coated microparticle swelling with a desirable delay. Coated WP/ALG microparticles appear to have potential as oral delivery systems for Saccharomyces boulardii or as encapsulation means for probiotic cells in pharmaceutical or food processing applications.     

Stabilization of pH-Induced Degradation of Porcine Insulin in Biodegradable Polyester Microspheres

The purpose of this research project was to stabilize the pH-induced degradation of porcine insulin encapsulated within biodegradable polyester microspheres through the incorporation of a basic additive. Insulin microspheres fabricated using Poly(L-lactide) (L-PLA) and Poly(DL-lactide-co-glycolide) (50:50 DL-PLGA) were subjected to in vitro release studies and the stability of unreleased insulin encapsulated within microspheres was investigated. The intramicrosphere pH was estimated by encapsulating acid-base indicators covering a wide pH transition range within 50:50 DL-PLGA microspheres. Finally, a basic excipient sodium bicarbonate was incorporated in 50:50 DL-PLGA microspheres to minimize acid-induced insulin degradation. The in vitro release was slow and incomplete (<30% in 30 days). Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of ～11% remained intact. The degradation products observed consisted of ～15% of three distinct deamidated hydrolysis products including A-21 Desamido insulin, ～22% Covalent Insulin Dimer and trace amounts of High Molecular Weight Transformation Products. Comparison of the degradation profile of unreleased insulin contained in various microsphere formulations with the in vitro release kinetics indicated that an increase in covalent dimer formation within the microspheres prior to release is associated with a decrease in the cumulative percent insulin released during a 30-day incubation period. In an attempt to correlate insulin degradation with the drop in intra-microsphere pH due to polymer hydrolysis, it was determined that the pH within a degrading microsphere reaches a value of ～1.8 after 4 weeks. The incorporation of a basic excipient, sodium bicarbonate, in 50:50 DL-PLGA microspheres resulted in an improved in vitro release profile (cumulative release ～47.3% in 30 days) as well as a significant reduction in covalent dimerization of the unreleased insulin to barely detectable levels. The low pH microenvironment within a degrading microsphere is one of the major factors leading to protein instability, and the degradation of proteins encapsulated within polyester microspheres can be minimized by the incorporation of a basic excipient.     

Preparation and testing of cefquinome-loaded poly lactic-co-glycolic acid microspheres for lung targeting

The aim of this study was to prepare cefquinome-loaded poly lactic-co-glycolic acid (PLGA) microspheres and to evaluate their in vitro and in vivo characteristics. Microspheres were prepared using a spry drier and were characterized in terms of morphology, size, drug-loading coefficient, encapsulation ratio and in vitro release. The prepared microspheres were spherical with smooth surfaces and uniform size (12.4?±?1.2?μm). The encapsulation efficiency and drug loading of cefquinome was 91.6?±?2.6 and 18.3?±?1.3%, respectively. In vitro release of cefquinome from the microspheres was sustained for 36?h. In vivo studies identified the lung as the target tissue and the region of maximum cefquinome release. A partial lung inflammation was observed but disappeared spontaneously as the microspheres were removed through in vivo decay. The sustained cefquinome release from the microspheres revealed its applicability as a drug delivery system that minimized exposure to healthy tissues while increasing the accumulation of therapeutic drug at the target site. These results indicated that the spray-drying method of loading cefquinome into PLGA microspheres is a straightforward method for lung targeting in animals.     

Sustained Polymeric Delivery of Gene Silencing Antisense ODNs,siRNA, DNAzymes and Ribozymes: In Vitro and In Vivo Studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (_d,_l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.     

红腺忍冬叶水提物抗氧化作用研究

摘要：目的 研究红腺忍冬叶水提物的抗氧化作用,为红腺忍冬叶的开发利用奠定科学依据。方法 通过对红腺忍冬叶水提醇沉法分离所得各部分的样品进行体内、体外抗氧化的实验研究,从而考察红腺忍冬叶水提物的抗氧化活性。以Vc、二丁基羟基甲苯（BHT）为对照品,通过DPPH法和铁离子还原法考察红腺忍冬叶不同样品的体外抗氧化活性。以Vc为阳性对照,由D-半乳糖（1g/kg）致衰的小鼠为动物模型,通过测定血清、肝脏组织中SOD、GSH-PX活性和MDA含量,考察样品的体内抗氧化活性。结果 红腺忍冬叶水提醇沉分离所得各部分体外抗氧化活性：在 0.1～5mg?mL-1范围内,90%乙醇直接沉淀物,30%乙醇沉淀物以及滤液部分具有较好的抑制DPPH自由基的活性和铁离子还原能力。体内SOD活性影响实验表明：30%乙醇沉淀物高剂量组、90%乙醇直接沉淀物低剂量组对提高小鼠体内SOD活性均有良好的效果,且都略优于Vc,但无显著性差异。体内GSH-PX活性影响实验表明：30%乙醇沉淀物高剂量组、90%乙醇直接沉淀物高剂量组和90%乙醇直接滤液低剂量组为佳。⑤体内MDA含量影响实验表明：30%乙醇沉淀物高、低剂量组和30%乙醇滤液高剂量组以及90%乙醇直接沉淀物低剂量组降低MDA含量的作用最为显著。结论 红腺忍冬叶水提液进一步醇沉分离所得各部分都具有不同程度的抗氧化效果,综合考虑以30%乙醇沉淀物活性最佳,值得进一步研究。     

pH triggered delivery of curcumin from Eudragit-coated chitosan microspheres for inflammatory bowel disease: characterization and pharmacodynamic evaluation

Objective: This investigation deals with the development and evaluation (in vitro and in vivo) of pH triggered Eudragit-coated chitosan microspheres of curcumin (CUR) for treating ulcerative colitis.

Methods: CUR-loaded chitosan microspheres were initially prepared by emulsion cross linking method followed by coating with Eudragit S-100. The pharmacodynamics of the developed formulation was analyzed in mice by acetic acid induced colitis model.

Results: The developed microspheres were of uniform spherical shape with high entrapment efficiency. CUR-chitosan microspheres showed less intense peaks compared to free CUR confirming inclusion of drug within microspheres as revealed by X-ray diffractogram. Uncoated CUR-chitosan microspheres exhibited burst release within initial 4?h while microspheres coated with Eudragit S-100 prevented premature release of CUR and showed controlled release up to 12?h following Higuchi model. In vivo organ biodistribution study showed negligible amount of CUR in stomach and small intestine confirming integrity of microsphere in upper gastrointestinal tract (GIT). In vivo study revealed significant reduction in severity and extent of colonic damage with CUR-loaded microspheres as compared to pure CUR which was further confirmed by histopathological study.

Conclusion: In vitro and in vivo studies proved the developed formulations as a promising system for pH-dependent delivery of drug to colon in ulcerative colitis.     

In Vivo and in Vitro Analysis of Skin Penetration Enhancement Based on a Two-Layer Diffusion Model with Polar and Nonpolar Routes in the Stratum Corneum

In vitro and in vivo skin penetration of three drugs with different lipophilicities and the enhancing effects of l-geranylazacycloheptan-2-one (GACH) were studied in rats. In vivo drug absorption profiles obtained by deconvolution of urinary excretion profiles were compared to the corresponding in vitro data obtained with a diffusion experiment. In vivo skin penetration of lipophilic butylparaben was considerably greater than that observed in vitro, while hydrophilic mannitol and acyclovir showed low penetration in both systems without GACH pretreatment. On the other hand, GACH enhanced mannitol and acyclovir penetration, especially in the in vivo system. Analysis of absorption profiles, using a two-layer skin model with polar and nonpolar routes in the stratum corneum, suggested that the diffusion length of a viable layer (viable epidermis and dermis) was shorter in vivo than in vitro and the effective area of the polar route in the stratum corneum was larger in vitro without GACH pretreatment. GACH increased the partitioning of acyclovir into the nonpolar route to the same extent in both systems. In addition, GACH increased the effective area of the polar route in vivo, probably because of enhanced water permeability; however, this effect was smaller in vitro since the stratum corneum was already hydrated even without GACH pretreatment.     

In vitro cytotoxicity, in vivo biodistribution and antitumor activity of HPMA copolymer–5-fluorouracil conjugates

5-Fluorouracil (5-FU) is an antimetabolite with a broad-spectrum activity against solid tumors. However, its very short half-life in plasma circulation greatly limited the in vivo antitumor efficacy and clinical application. The current work aimed to solve this problem as well as to increase 5-FU biodistribution to tumor by covalently conjugating 5-FU to a biocompatible, non-toxic and non-immunogenic drug carrier – N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer. The in vitro cytotoxicity, in vivo biodistribution and therapeutic efficacy of HPMA copolymer–5-FU conjugates (P-FU) were reported. Cytotoxicity was evaluated by using a serial of tumor cells (A549, CT-26, Hela, HepG₂ cells and 5-FU resistant HepG₂ cells). In vivo biodistribution and therapeutic efficacy were investigated in Kunming mice-bearing hepatoma 22 (H₂₂). Results indicated that P-FU could increase the cytotoxicity of 5-FU in Hela, HepG₂ and 5-FU resistant HepG₂ cells, while it decreases the cytotoxicity of 5-FU in A549 and CT-26. Both in vitro release profile in plasma and biodistribution study showed that P-FU significantly prolonged the drug plasma circulation time. P-FU also showed an over 3-fold larger area under the concentration–time curve (AUC) in tumor when compared with free drug. Therapeutic evaluation also demonstrated that the treatment with P-FU displayed stronger inhibition of the tumor growth when compared with that of control group (physiologic saline) or 5-FU group at the same dose. All the results suggested that P-FU could increase cytotoxicity of 5-FU in certain cancer cell lines, prolong 5-FU circulation time in vivo, enhance 5-FU distribution to tumor and improve therapeutic efficacy. Therefore, HPMA copolymer is a potential carrier for 5-FU for the effective treatment of cancer.     

肺靶向卡铂明胶微球的研究

目的:为提高卡铂的疗效,降低毒副作用,制备了该药的明胶微球。方法:用乳化法制备卡铂明胶微球,紫外分光光度法测定药物的含量,二阶导数法测定体外释药情况;用静脉注射肿瘤细胞建立了肺肿瘤模型,计算瘤结节数来考察疗效。结果:卡铂明胶微球平均粒径为13-20 μm ,粒径范围5-0～28-6 μm 的微球数占总数的91-8% 。微球平均载药量为23-76%(n=3) 。冰箱、室温和37℃,RH75% 考察3个月,几乎无变化,体外释药符合一级动力学规律,释药T_1/2 比原药延长约10倍。药效学实验表明,卡铂明胶微球对小鼠肺部S180肿瘤生长有明显的抑制作用,抑瘤作用较原药卡铂大大提高。结论:卡铂明胶微球在体内有良好的肺靶向性,对提高药物的疗效,降低药物毒副作用等方面有重大意义。     

Targeting microparticles to select tissue via radiation-induced upregulation of endothelial cell adhesion molecules

Purpose. Certain endothelial cell adhesion molecules are up regulated in tissue that has been irradiated for therapeutic purposes. This up-regulation of adhesion molecules provides a potential avenue for targeting drugs to select tissues. Methods. Microspheres were coated with a mAb to ICAM-1 and the level of adhesion of the anti-ICAM-1 microspheres to irradiated tissue in vitro and in vivo was quantified. Results. Under in vitro flow conditions, the number of adherent microspheres on irradiated HUVEC was 4.8 ± 0.9 times that of control; the adhesion of anti-ICAM-1 microspheres on irradiated HUVEC could be enhanced by more than 170% in the presence of RBC (20% hematocrit) in the medium. In vivo in a rat cranial window model, the number of adherent anti-ICAM-1 microspheres in locally irradiated cerebral tissue was 8 and 13 times that of IgG microspheres at 24 h and 48 h post-irradiation, respectively and returned to baseline 7 days post-irradiation. In locally irradiated animals, the number of adhering microspheres in unirradiated tissue remained at the basal level. Conclusions. Radiation-induced up-regulation of endothelial cell adhesion molecules may be exploited to target drugs and/or genes to select segments of the endothelium.     

Enhancing and sustaining the topical ocular delivery of fluconazole using chitosan solution and poloxamer/chitosan in situ forming gel

Fungal keratitis is a serious disease that can lead to loss of vision. Unfortunately, current therapeutic options often result in poor bioavailability of antifungal agents due to protective mechanisms of the eye. The aim of this work was to evaluate the potential of a chitosan solution as well as an in situ gel-forming system comprised of poloxamer/chitosan as vehicles for enhanced corneal permeation and sustained release of fluconazole (FLU). For this, in vitro release and ex vivo corneal permeation experiments were carried out as a function of chitosan concentration from formulation containing the chitosan alone and combined with the thermosensitive polymer, poloxamer. Microdialysis was employed in a rabbit model to evaluate the in vivo performance of the formulations. The in vitro release studies showed the sustained release of FLU from the poloxamer/chitosan formulation. Ex vivo permeation studies across porcine cornea demonstrated that the formulations studied have a permeation-enhancing effect that is independent of chitosan concentration in the range from 0.5 to 1.5% w/w. The chitosan solutions alone showed the greatest ex vivo drug permeation; however, the poloxamer/chitosan formulation presented similar in vivo performance than the chitosan solution at 1.0%; both formulations showed sustained release and about 3.5-fold greater total amount of FLU permeated when compared to simple aqueous solutions of the drug. In conclusion, it was demonstrated that both the in situ gelling formulation evaluated and the chitosan solution are viable alternatives to enhance ocular bioavailability in the treatment of fungal keratitis.     

Formulation and evaluation of stomach-specific amoxicillin-loaded carbopol-934P mucoadhesive microspheres for anti-Helicobacter pylori therapy

The purpose of this research was to formulate and systemically evaluate in vitro and in vivo performances of mucoadhesive amoxicillin microspheres for the potential use in the treatment of gastric and duodenal ulcers, which were associated with Helicobacter pylori. Amoxicillin mucoadhesive microspheres containing carbopol-934P as mucoadhesive polymer and ethyl cellulose as carrier polymer were prepared by an emulsion-solvent evaporation technique. Results of preliminary trials indicate that quantity of emulsifying agent, time for stirring, drug-to-polymers ratio and speed of rotation affected the characteristics of microspheres. Microspheres were discrete, spherical, free flowing and showed a good percentage of drug entrapment efficiency. An in vitro mucoadhesive test showed that amoxicillin mucoadhesive microspheres adhered more strongly to the gastric mucous layer and could retain in the gastrointestinal tract for an extended period of time. A 3² full factorial design was employed to study the effect of independent variables, drug-to-polymer-to-polymer ratio (amoxicillin-ethyl cellulose-carbopol-934P) (X₁) and stirring speed (X₂) on dependent variables, i.e. percentage mucoadhesion, drug entrapment efficiency, particle size and t₈₀. The best batch exhibited a high drug entrapment efficiency of 56%; mucoadhesion percentage after 1 h was 80% and the particle size was 109 µm. A sustained drug release was obtained for more than 12 h. The drug-to-polymer-to-polymer ratio had a more significant effect on the dependent variables. The morphological characteristics of the mucoadhesive microspheres were studied under a scanning electron microscope. In vitro release test showed that amoxicillin released slightly faster in pH 1.2 hydrochloric acid than in pH 7.8 phosphate buffer. In vivo H. pylori clearance tests were also carried out by administering amoxicillin powder and mucoadhesive microspheres to H. pylori infectious Wistar rats under fed conditions at single dose or multiple dose(s) in oral administration. The results showed that amoxicillin mucoadhesive microspheres had a better clearance effect than amoxicillin powder. In conclusion, the prolonged gastrointestinal residence time and enhanced amoxicillin stability resulting from the mucoadhesive microspheres of amoxicillin might make a contribution to H. pylori complete eradication.