Comparison between Colistin Sulfate Dry Powder and Solution for Pulmonary Delivery.

To assess the difference in the fate of the antibiotic colistin (COLI) after its pulmonary delivery as a powder or a solution, we developed a COLI powder and evaluated the COLI pharmacokinetic properties in rats after pulmonary administration of the powder or the solution. The amorphous COLI powder prepared by spray drying was characterized by a mass median aerodynamic diameter and fine particle fraction of 2.68 ± 0.07 µm and 59.5 ± 5.4%, respectively, when emitted from a Handihaler®. After intratracheal administration, the average pulmonary epithelial lining fluid (ELF): plasma area under the concentration versus time curves (AUC) ratios were 570 and 95 for the COLI solution and powder, respectively. However, the same COLI plasma concentration profiles were obtained with the two formulations. According to our pharmacokinetic model, this difference in ELF COLI concentration could be due to faster systemic absorption of COLI after the powder inhalation than for the solution. In addition, the COLI apparent permeability (Papp) across a Calu-3 epithelium model increased 10-fold when its concentration changed from 100 to 4000 mg/L. Based on this last result, we propose that the difference observed in vivo between the COLI solution and powder could be due to a high local ELF COLI concentration being obtained at the site where the dry particles impact the lung. This high local COLI concentration can lead to a local increase in COLI Papp, which is associated with a high concentration gradient and could produce a high local transfer of COLI across the epithelium and a consequent increase in the overall absorption rate of COLI.

Sustained-release microparticle dry powders of chloramphenicol palmitate or thiamphenicol palmitate prodrugs for lung delivery as aerosols.

The purpose of this study was to design inhalable sustained-release nanoparticle-in-microparticles, i.e. nano-embedded microparticles, for the lung delivery of chloramphenicol or thiamphenicol as aerosols. The palmitate ester prodrugs of the two antibiotics were used to prepare PLGA-based nanoparticles or to form pure prodrug nanoparticles. Prodrug-loaded PLGA nanoparticles or pure prodrug nanoparticles were prepared using the emulsion-solvent evaporation method. Dry microparticle powders for inhalation were then produced by spray-drying the nanoparticle suspensions supplemented with lactose as a bulking agent and L-leucine as a dispersing enhancer. Examined under the scanning electron microscopy, the obtained microparticles appeared to be spherical and shriveled, with no crystal-like structures. Drug loading was satisfactory (14 to 34% (m/m)) and the aerodynamic properties determined with a Next Generation Impactor were appropriate for lung delivery, with mass median aerodynamic diameters close to 3 µm. The in vitro release profiles showed that sustained released was achieved with these formulations, with an almost complete release over 14 days.

Development of levofloxacin-loaded PLGA microspheres of suitable properties for sustained pulmonary release.

Aerosol antibiotics are an interesting alternative to oral or intravenous therapy in Cystic Fibrosis lung infections. Levofloxacin (LVX) inhaled solution is already an effective option. In this study, the aim was the development of LVX-loaded PLGA microspheres (MS) for pulmonary administration as a dry powder. MS were prepared, for the first time, by a modified double emulsion solvent evaporation method with premix membrane homogenization. Aqueous phases were saturated with LVX and a fatty acid (lauric acid) was added to avoid the drug escaping from the organic phase. MS were characterized in terms of size, drug content, morphology and in vitro release properties. X-ray diffraction, Fourier-transform infrared spectroscopy, differential and gravimetric thermal analysis, and cytotoxicity analyses were performed. Results showed this new method increased the drug loading while maintaining an adequate (∼5 µm) particle size and controlled release. Compared to a solution for inhalation, these properties combined with the dry-powder nature of these MS will improve patient compliance. The incorporation of lauric acid was not advantageous because the particle size was higher and no improvements concerning the sustained release occurred. LVX was molecularly dispersed in the matrix, or it was in amorphous state, as confirmed by the physico-chemical analyses. Calu-3 cell viability assays demonstrated no cytotoxicity for these MS, making them a promising system for LVX pulmonary delivery.

Active Mediated Transport of Chloramphenicol and Thiamphenicol in a Calu-3 Lung Epithelial Cell Model.

Pulmonary administration enables high local concentrations along with limited systemic side effects but not all antibiotics could be good candidates. In this perspective, diffusion of the antibiotic chloramphenicol (CHL) and thiamphenicol (THA) through the lung has been evaluated to reassess their potential for pulmonary administration. The apparent permeability (Papp) was evaluated with the Calu-3 cell model. The influence of drug transporters was assessed with the PSC-833, MK-571, and KO-143 inhibitors. The influence of CHL and THA on the cell uptake of rhodamin 123 and fluorescein was also evaluated. Absorptive Papp of CHL and THA was concentration independent with CHL Papp 4 times higher than that of THA. Secretory Papp of CHL was concentration independent, whereas it was concentration dependent for THA with an efflux ratio of 3.6 for the lowest concentration. The use of inhibitors suggested that CHL and THA were substrates of efflux transporters but with a low affinity. In conclusion, the permeability results suggest that the pulmonary route may offer a biopharmaceutical advantage only for THA. Owing to the influence of drug transporters, a higher concentration in the lung than in the plasma is expected mostly for THA, whatever the route of administration.

Pulmonary pharmacokinetics of levofloxacin in rats after aerosolization of immediate-release chitosan or sustained-release PLGA microspheres.

A comparative pharmacokinetic study was conducted in rats after intratracheal aerosolization of levofloxacin, as a solution, as immediate-release chitosan microspheres or as sustained-release PLGA microspheres. A pharmacokinetic model was constructed to model levofloxacin concentrations both in plasma and in the lung epithelial lining fluid (ELF). The plasma and ELF experimental concentration profiles versus time were similar for the intravenous and intratracheal levofloxacin solutions and for the intratracheal levofloxacin-loaded chitosan microsphere dry powder, indicating that levofloxacin diffused almost instantaneously through the broncho-alveolar barrier and that the chitosan microspheres released levofloxacin very rapidly, as anticipated from in vitro release studies. The bioavailability for the intratracheal levofloxacin solution and intratracheal chitosan microspheres was estimated to be 98% and 71%, respectively, both with a direct release into the ELF compartment. The ELF-to-unbound plasma AUC ratios were slightly above 2 and may result from an efflux transport. For the intratracheal PLGA microspheres, a high ELF-to-unbound plasma AUC concentration ratio (311) was observed and high levofloxacin concentrations were maintained in ELF for at least 72h in consistency with the in vitro release studies. The bioavailability was 92%, with 19% of the dose released immediately (burst release) into the ELF and 73% released slowly into the ELF from a depot compartment, i.e. the PLGA microspheres, according to a Weibull model. These results highlight the benefit of using sustained-release microspheres administered as aerosols to provide and to maintain high pulmonary concentrations of an antibiotic characterized with a high permeability profile through the broncho-alveolar barrier. The sustained-release microsphere dry powder aerosol may therefore provide advantages over solutions or pure drug dry powders for inhalation in terms of treatment efficiency, ease of use and frequency of administration.

Ciprofloxacin-Loaded Inorganic-Organic Composite Microparticles To Treat Bacterial Lung Infection.

Ciprofloxacin (CIP) is an antibiotic that has been clinically trialed for the treatment of lung infections by aerosolization. However, CIP is rapidly systemically absorbed after lung administration, increasing the risk for subtherapeutic pulmonary concentrations and resistant bacteria selection. In the presence of calcium, CIP forms complexes that reduce its oral absorption. Such complexation may slow down CIP absorption from the lung thereby maintaining high concentration in this tissue. Thus, we developed inhalable calcium-based inorganic-organic composite microparticles to sustain CIP within the lung. The aerodynamics and micromeritic properties of the microparticles were characterized. FTIR and XRD analysis suggest that the inorganic component of the particles comprised amorphous calcium carbonate and amorphous calcium formate, and that CIP and calcium interact in a 1:1 stoichiometry in the particles. CIP was completely released from the microparticles within 7 h, with profiles showing a slight dependence on pH (5 and 7.4) compared to the dissolution of pure CIP. Transport studies of CIP across Calu-3 cell monolayers, in the presence of various calcium concentrations, showed a decrease of up to 84% in CIP apparent permeability. The apparent minimum inhibitory concentration of CIP against Pseudomonas aeruginosa and Staphylococcus aureus was not changed in the presence of the same calcium concentration. These results indicate that the designed particles should provide sustained levels of CIP with therapeutic effect in the lung. With these microparticles, it should be possible to control CIP pharmacokinetics within the lung, based on controlled CIP release from the particles and reduced apparent permeability across the epithelial barrier due to the cation-CIP interaction.

Optimization of levofloxacin-loaded crosslinked chitosan microspheres for inhaled aerosol therapy.

The aim of this work was the development of innovative levofloxacin-loaded swellable microspheres (MS) for the dry aerosol therapy of pulmonary chronicPseudomonas aeruginosainfections in Cystic Fibrosis patients. In a first step, a factorial design was applied to optimize formulations of chitosan-based MS with glutaraldehyde as crosslinker. After optimization, other crosslinkers (genipin, glutaric acid and glyceraldehyde) were tested. Analyses of MS included aerodynamic and swelling properties, morphology, drug loading, thermal and chemical characteristics,in vitroantibacterial activity and drug release studies. The prepared MS presented a drug content ranging from 39.8% to 50.8% of levofloxacin in an amorphous or dispersed state, antibacterial activity and fast release profiles. The highest degree of swelling was obtained for MS crosslinked with glutaric acid and genipin. These formulations also presented satisfactory aerodynamic properties, making them a promising alternative, in dry-powder inhalers, to levofloxacin solution for inhalation.

Passive and active strategies for transdermal delivery using co-encapsulating nanostructured lipid carriers: in vitro vs. in vivo studies.

This work aimed at designing a formulation based on nanostructured lipid carriers (NLC) for transdermal co-administration of olanzapine and simvastatin, using passive and active strategies in a combined in vitro/in vivo development approach. NLC were prepared by two distinct methods, namely solvent emulsification-evaporation (SE/E) and high pressure homogenization (HPH). HPH was selected on the basis of a better performance in terms of drug loading and in vitro permeation rate. Several mathematical models were used to elucidate the release mechanisms from lipid nanoparticles. In vitro release kinetics was shown to be driven by diffusion, but other mechanisms were also present, and supported the feasibility of using NLC for sustained drug delivery. The in vitro skin studies showed that the chemical penetration enhancers, limonene and ethanol, added to the NLC formulations, promoted a synergistic permeation enhancement of both drugs, with olanzapine exhibiting a higher permeation than simvastatin. Transdermal administration to rats resulted in steady-state levels reached at around 10h and maintained for 48h, again with olanzapine exhibiting a better permeation rate. The pharmacokinetic parameters indicated that the NLC dispersion displayed a better in vivo performance than the gel, which was consistent with the in vitro results. These differences were, however, negligible in the flux values, supporting the use of gel as a final, more convenient, formulation. The in vivo experiments in rats correlated well with in vitro findings and revealed that the combined use of ethanol and limonene, incorporated in the NLC formulation, provided the main driving force for drug permeation. The Dermaroller® pretreatment did not significantly enhance drug permeation, supporting the use of passive methods as suitable for a transdermal delivery system. Furthermore, this work may provide a promising proof-of-concept for further clinical application in the treatment of schizophrenia and associated disorders, combined with dyslipidemia.

Aerosol therapy with colistin methanesulfonate: a biopharmaceutical issue illustrated in rats.

The aim of this study was to evaluate the biopharmaceutical behavior of colistin methanesulfonate (CMS) with special focus on colistin presystemic formation after CMS nebulization in rats. CMS was administered (15 mg x kg(-1) of body weight) either intravenously for systemic pharmacokinetic studies (n = 6) or as an intratracheal nebulization for systemic pharmacokinetic studies (n = 5) or for CMS and colistin concentration measurements in epithelial lining fluid (ELF) at 30, 120, and 240 min after nebulization (n = 14). CMS and colistin concentrations were determined by a new liquid chromatography (LC)-tandem mass spectrometry (MS/MS) assay. Pharmacokinetic parameters were estimated by noncompartmental analysis. CMS and colistin pharmacokinetic data were consistent with previously published values when comparisons were possible. The fraction of the CMS dose converted systematically into colistin after intravenous CMS administration was estimated to be 12.5% on average. After CMS nebulization it was estimated that about two-thirds of the dose was directly absorbed within the systemic circulation, whereas one-third was first converted into active colistin, which was eventually absorbed. As a consequence, the colistin area under curve (AUC) reflecting systemic availability was about 4-fold greater after CMS intratracheal nebulization (607 +/- 240 microg x min x ml(-1)) than after CMS intravenous administration (160 +/- 20 microg x min x ml(-1)). CMS concentrations in ELF at 30 min and 120 min postnebulization were very high (in the order of several mg/ml) due to the limited volume of ELF but were considerably reduced at 240 min. Although lower (15% +/- 5% at 120 min) in relative terms, colistin concentrations in ELF could be high enough for being active against microorganisms following CMS nebulization.

Assay of colistin and colistin methanesulfonate in plasma and urine by liquid chromatography-tandem mass spectrometry.

A rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the routine quantification of colistins A and B and their prodrugs, colistin methanesulfonate (CMS) A and CMS B, respectively, in human plasma and urine by using polymyxin B1 as the internal standard (IS). CMS concentrations were determined indirectly by subtracting the colistin concentrations determined in biological samples from the whole colistin concentrations determined after sample treatment with sulfuric acid in order to hydrolyze CMS into colistin. After extraction on a solid-phase extraction column, the colistins were separated on an XBrigde C(18) column with isocratic elution (run time, 3.8 min). The mobile phase was 0.1% (vol/vol) formic acid in acetonitrile-0.1% (vol/vol) formic acid in water (20:80, vol/vol), run at a 0.2-ml/min flow rate. Ions were detected in the turbo-ion-spray-positive and multiple-reaction-monitoring modes. The ions monitored (precursor [M + 2H](2+) to product ions) were m/z 585.5/101.2 for colistin A, m/z 578.5/101.2 for colistin B, and m/z 602.5/241.2 for IS. Prevalidation studies demonstrated the stability of CMS in biological samples and extracts, a key point for the reliable quantification of colistin and CMS. The assay was accurate and reproducible for the quantification of colistins A and B and CMSs A and B in plasma samples over concentration ranges appropriate for pharmacokinetic studies: 0.024 to 6.144, 0.015 to 3.856, 0.029 to 7.492, and 0.010 to 2.508 microg/ml, respectively. In urine samples, the assay was validated over the same concentration ranges for colistins and over concentration ranges of 0.058 to 7.492 microg/ml and 0.020 to 2.508 microg/ml for CMSs A and B, respectively.

P-glycoprotein-mediated transport of moxifloxacin in a Calu-3 lung epithelial cell model.

Moxifloxacin (MXF) is a fluoroquinolone antibiotic that is effective against respiratory infections. However, the mechanisms of MXF lung diffusion are unknown. Active transport in other tissues has been suggested for several members of the fluoroquinolone family. In this study, transport of MXF was systematically investigated across a Calu-3 lung epithelial cell model. MXF showed polarized transport, with the secretory permeability being twice as high as the absorptive permeability. The secretory permeability was concentration dependent (apparent P(max) = 13.6 x 10(-6) cm x s(-1); apparent K(m) = 147 microM), suggesting saturated transport at concentrations higher than 350 microg/ml. The P-glycoprotein inhibitor PSC-833 inhibited MXF transport in both directions, whereas probenecid, a multidrug resistance-related protein inhibitor, appeared to have no effect in the Calu-3 model. Moreover, rifampin, a known inducer of efflux transport proteins, upregulated the expression of P-glycoprotein in Calu-3 cells and enhanced MXF active transport. In conclusion, this study clearly indicates that MXF is subject to P-glycoprotein-mediated active transport in the Calu-3 model. This P-glycoprotein-dependent secretion may lead to higher MXF epithelial lining fluid concentrations than those in plasma. Furthermore, drug-drug interactions may be expected when MXF is combined with other P-glycoprotein substrates or modulators.

Formulation of rifampicin-cyclodextrin complexes for lung nebulization.

Lung administration of antibiotics by nebulization is promising for improving treatment efficiency for pulmonary infections, as it increases drug concentration at sites of infection while minimizing systemic side effects. For poorly soluble molecules like rifampicin, cyclodextrins (CD) may improve lung delivery by permitting higher dosing. For this purpose, we investigated rifampicin-CD complexes in terms of rifampicin apparent solubility enhancement, effect on in vitro permeability on Calu-3 broncho-alveolar cells, effect on in vitro antibacterial activity against Acinetobacter baumannii and nebulization characteristics measured by NGI cascade impactor. Complexation efficiency between rifampicin and methylated beta-cyclodextrin (RAMEB) or hydroxypropyl-beta-cyclodextrin (HPbetaCD) was pH-dependent, involving the piperazin group. Rifampicin phase solubility diagrams constructed at pH 9 showed an A(L)-type curve for RAMEB and a B(S)-type for HPbetaCD. Stability constants calculated for a 1:1 molar ratio of CD/rifampicin were 73.4 +/- 8.2 M(-1) for RAMEB and 68.5 +/- 5.2 M(-1) for HPbetaCD. Complexes with RAMEB or HPbetaCD increased 22 times and 7.6 times respectively the apparent solubility of rifampicin and were found to be satisfactorily stable for 2 days when diluted in a solution at physiological pH. The nebulization of the complex solution created droplets in size range compatible with pulmonary deposition. Furthermore, the presence of HPbetaCD decreased the MMAD of the aerosolized droplets. Activity of RAMEB and HPbetaCD complexes measured by the total rifampicin MIC against A. baumannii was similar or lower to free rifampicin MIC respectively. Complexation did not alter the rifampicin permeability in the timescale of 1h as evaluated with a Calu-3 epithelial cell model, but acted as a reservoir for rifampicin. In conclusion, this work reports that CDs can be used as vectors for pulmonary nebulization to increase the amount of active rifampicin and optimize its lung pharmacokinetic profile.

LC-MS/MS determination of the HIV-1 protease inhibitor indinavir in brain and testis of mice.

A rapid and sensitive method for the determination of indinavir in mice brain and testis is described and validation data are provided. Indinavir and the internal standard (IS) amprenavir were isolated from homogenized tissue matrices using a mixed-mode solid-phase extraction (SPE) procedure and were then analyzed by reversed-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS). The mass spectrometer in the positive-ion multiple reaction monitoring mode used pairs of ions at m/z of 614.1/421.3 for indinavir and of 506.1/245.3 for IS. The calibration curves were linear over the range 0.0012-0.0390 micromol/kg for brain and 0.39-12.50 micromol/kg for testis. Linearity, repeatability and accuracy were validated. The applicability of the method was demonstrated by assessing indinavir in brain and testis of three mice dosed with intravenous bolus administration of indinavir (16.3 micromol/kg).

Tissue distribution of indinavir administered as solid lipid nanocapsule formulation in mdr1a (+/+) and mdr1a (-/-) CF-1 mice.

PURPOSE: Due to protease inhibitor (PI) efflux transport by P-glycoprotein (P-gp), insufficient PI concentrations result in low ongoing HIV replication in the so-called virus sanctuaries (brain and testes). The aim of the present study was to evaluate indinavir-loaded nanocapsules (Ind-LNC) including Solutol HS15, an excipient reported to possess in vitro P-gp inhibiting properties, as a means to improve indinavir distribution into brain and testes of mice. METHODS: Normal mdr1a (+/+) or P-gp-deficient mdr1a (-/-) CF-1 mice were dosed with Ind-LNC (10 mg indinavir/kg, i.v.). At 30 min post-administration, indinavir was determined in plasma, brain, testes, as well as in kidneys, liver, and heart by LC-MS/MS, and tissue/plasma concentration ratios were calculated. Results were compared with those of control groups that received an indinavir solution (Ind-Sol). RESULTS: Using Ind-Sol, ratios were 21.3- and 3.3-fold higher in brains and testes of mdr1a (-/-) mice than of mdr1a (+/+) mice, respectively, whereas in the other organs ratios were not significantly different between the two substrains. When Ind-LNC was used, a similar [mdr1a(-/-) vs. mdr1a (+/+) mice] trend was observed. Moreover, ratios were found to be significantly increased (1.9-fold increase in average) in most organs (brain and testes in particular) with Ind-LNC compared to Ind-Sol, regardless of the substrain used. CONCLUSIONS: In agreement with previous works, P-gp governs at least in part indinavir uptake into brain and testes. LNC formulation increased indinavir uptake in brain and testes by mechanisms other than, or additional to, P-gp inhibition.

Investigation of oral bioavailability and brain distribution of the Ind(8)-Val conjugate of indinavir in rodents.

Protease inhibitors are successfully used for the treatment of acquired immune deficiency syndrome (AIDS) although their biopharmaceutical characteristics are not optimal. Prodrugs have therefore been synthesized to increase protease inhibitor bioavailability and brain distribution. Among several compounds tested, a valine derivative of indinavir (Ind(8)-Val) showed promising characteristics using an in-vitro Caco-2 cell model. The objective of this study was to further investigate this compound using in-situ and in-vivo approaches. The pharmacokinetics of indinavir (Ind) and Ind(8)-Val were investigated in rats after intravenous and oral administration. Free indinavir resulting from in-vivo hydrolysis of Ind(8)-Val could not be detected in the plasma of rats receiving Ind(8)-Val. Furthermore Ind(8)-Val bioavailability was only 32% on average compared with 76% for indinavir, and effective permeability coefficients determined with a single-pass intestinal perfusion method were close to 25x10(6)cms(-1) for the two compounds. Brain-to-plasma concentration ratios in the post equilibrium phase after intravenous administration to mice were 9.7+/-8.1% for indinavir and 2.5+/-2.7% for Ind(8)-Val. In conclusion, the promising biopharmaceutical characteristics of Ind(8)-Val suggested from previous in-vitro experiments with the Caco-2 cell model were not confirmed by in-situ and in-vivo experiments.

Drug transport to brain with targeted nanoparticles.

Nanoparticle drug carriers consist of solid biodegradable particles in size ranging from 10 to 1000 nm (50-300 nm generally). They cannot freely diffuse through the blood-brain barrier (BBB) and require receptor-mediated transport through brain capillary endothelium to deliver their content into the brain parenchyma. Polysorbate 80-coated polybutylcyanoacrylate nanoparticles can deliver drugs to the brain by a still debated mechanism. Despite interesting results these nanoparticles have limitations, discussed in this review, that may preclude, or at least limit, their potential clinical applications. Long-circulating nanoparticles made of methoxypoly(ethylene glycol)- polylactide or poly(lactide-co-glycolide) (mPEG-PLA/PLGA) have a good safety profiles and provide drug-sustained release. The availability of functionalized PEG-PLA permits to prepare target-specific nanoparticles by conjugation of cell surface ligand. Using peptidomimetic antibodies to BBB transcytosis receptor, brain-targeted pegylated immunonanoparticles can now be synthesized that should make possible the delivery of entrapped actives into the brain parenchyma without inducing BBB permeability alteration. This review presents their general properties (structure, loading capacity, pharmacokinetics) and currently available methods for immunonanoparticle preparation.

Glucose-targeted niosomes deliver vasoactive intestinal peptide (VIP) to the brain.

The aim of this study was to evaluate glucose-bearing niosomes as a brain targeted delivery system for the vasoactive intestinal peptide (VIP). To this end, VIP/125I-VIP-loaded glucose-bearing niosomes were intravenously injected to mice. Brain uptake was determined by measuring the radioactivity of 125I-labeled VIP using gamma-counting, after intravenous administration of VIP in solution or encapsulated in glucose-bearing niosomes or in control niosomes. VIP integrity was assessed by reversed-phase HPLC analysis of brain extracts. Distribution of 125I-VIP derived radioactivity was examined from serial brain slices. HPLC analysis confirmed the presence of intact VIP in brain after administration of VIP-loaded niosomes, but not after administration of VIP solution. Encapsulation within glucose-bearing niosomes mainly allowed a significantly higher VIP brain uptake compared to control niosomes (up to 86%, 5min after treatment). Brain distribution of intact VIP after injection of glucose-bearing niosomes, indicated that radioactivity was preferentially located in the posterior and the anterior parts of the brain, whereas it was homogeneously distributed in the whole brain after the administration of control vesicles. In conclusion, this novel vesicular formulation of VIP delivers intact VIP to particular brain regions in mice. Glucose-bearing vesicles might be therefore a novel tool to deliver drugs across the blood-brain barrier (BBB).

Glucose-receptor MR imaging of tumors: study in mice with PEGylated paramagnetic niosomes.

PURPOSE: To evaluate a magnetic resonance (MR) imaging contrast agent for tumor detection based on paramagnetic nonionic vesicles (niosomes) bearing polyethylene glycol (PEG) and glucose conjugates for the targeting of overexpressed glucose receptors. MATERIALS AND METHODS: Four gadobenate dimeglumine-loaded niosome preparations including nonconjugated niosomes, niosomes bearing glucose conjugates (N-palmitoyl glucosamine [NPG]), niosomes bearing PEG 4400, and niosomes bearing both PEG and NPG were tested. In vitro cellular uptake was measured at electron paramagnetic resonance (EPR) after incubation with human prostate carcinoma, PC3, cells. In vivo distribution was studied at MR imaging 6, 12, and 24 hours after injection, with assessment of tumor, brain, liver, and muscle signal intensity (SI) in 49 mice bearing PC3 cells. Efficiency of targeted contrast agents was assessed with tumor-to-muscle contrast-to-noise ratio (CNR). Testing for differences was performed with analysis of variance followed by a posteriori Fisher test. RESULTS: In vitro, gadolinium could be detected at EPR only in cell pellets incubated with niosomes bearing glucose conjugates or niosomes bearing both glucose conjugates and PEG (4.9. 10(-15) and 4.5. 10(-15) mol gadolinium per PC3 cell). In vivo, marked predominant tumor enhancement was demonstrated 24 hours after injection of glycosylated PEG niosomes (P <.01); no significant differences were observed following injection of nonconjugated niosomes, glycosylated niosomes, or PEG 4400 niosomes. Twenty-four hours after injection, sole presence of NPG or PEG 4400 on the surface of the niosome led to higher tumor-to-muscle CNR than that observed after injection of nonconjugated niosomes (CNR of 3.3 +/- 0.7 [SD], 3.4 +/- 2.2, and 0 +/- 1.9). Combination of NPG and PEG led to even higher tumor-to-muscle CNR (6.3 +/- 2.2). CONCLUSION: Combination of PEG and glucose conjugates on the surface of niosomes significantly improved tumor targeting of an encapsulated paramagnetic agent assessed with MR imaging in a human carcinoma xenograft model.

Anticancer drug delivery with transferrin targeted polymeric chitosan vesicles.

PURPOSE: The study reports the initial biological evaluation of targeted polymeric glycol chitosan vesicles as carrier systems for doxorubicin (Dox). METHODS: Transferrin (Tf) was covalently bound to the Dox-loaded palmitoylated glycol chitosan (GCP) vesicles using dimethylsuberimidate (DMSI). For comparison, glucose targeted niosomes were prepared using N-palmitoyl glucosamine. Biological properties were studied using confocal microscopy, flow cytometry, and cytotoxicity assays as well as a mouse xenograft model. RESULTS: Tf vesicles were taken up rapidly with a plateau after 1-2 h and Dox reached the nucleus after 60-90 min. Uptake was not increased with the use of glucose ligands, but higher uptake and increased cytotoxicity were observed for Tf targeted as compared to GCP Dox alone. In the drug-resistant A2780AD cells and in A431 cells, the relative increase in activity was significantly higher for the Tf-GCP vesicles than would have been expected from the uptake studies. All vesicle formulations had a superior in vivo safety profile compared to the free drug. CONCLUSIONS: The in vitro advantage of targeted Tf vesicles did not translate into a therapeutic advantage in vivo. All vesicles reduced tumor size on day 2 but were overall less active than the free drug.

Effects of the vasoactive intestinal peptide (VIP) and related peptides on glioblastoma cell growth in vitro.

The growth rate of numerous cancer cell lines is regulated in part by actions of neuropeptides of the vasoactive intestinal peptide (VIP) family, which also includes pituitary adenylate cyclase-activating peptide (PACAP), glucagon, and peptide histidine/isoleucine (PHI). The aim of this work was to investigate the effect of these peptides on the growth of the rat glioblastoma cell line C6 in vitro. We also sought to determine which binding sites were correlated with the effects observed. Proliferation studies performed by means of a CyQuant trade mark assay showed that VIP and PACAP strongly stimulated C6 cell proliferation at most of the concentrations tested, whereas PHI increased cell proliferation only when associated with VIP. Two growth hormone-releasing factor (GRF) derivatives and the VIP antagonist hybrid peptide neurotensin-VIP were able to inhibit VIP-induced cell growth stimulation, even at very low concentrations. Binding experiments carried out on intact cultured C6 cells, using 125I-labeled VIP and PACAP as tracers, revealed that the effects of the peptides on cell growth were correlated with the expression on C6 cells of polyvalent high-affinity VIP-PACAP binding sites and of a second subtype corresponding to very high-affinity VIP-selective binding species. The latter subtype, which interacted poorly with PACAP with a 10,000-fold lower affinity than VIP, might mediate the antagonist effects of neurotensin- VIP and of both GRF derivatives on VIP-induced cell growth stimulation.