Article

Kinetic Analysis of the Toxicity of Pharmaceutical Excipients Cremophor EL and RH40 on Endothelial and Epithelial Cells

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Cremophor EL and RH40 are widely used excipients in oral and intravenous drug formulations such as Taxol infusion to improve drug dissolution and absorption. Studies indicate that Cremophors, especially EL, have toxic side effects, but few data are available on endothelial and epithelial cells, which form biological barriers and are directly exposed to these molecules. Human hCMEC/D3 brain endothelial and Caco-2 epithelial cells were treated with Cremophor EL and RH40 in the 0.1-50 mg/mL concentration range. Cell toxicity was monitored by real-time cell microelectronic sensing and verified by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and morphological methods. Cremophors caused dose- and time-dependent damage in both cell types. In endothelial cells, 0.1 mg/mL and higher concentrations, in epithelial cells, concentrations of 5 mg/mL and above were toxic, especially at longer incubations. Cell death was also proven by double fluorescent staining of cell nuclei. Immunostaining for tight junction proteins claudin-4 and -5 showed barrier disruption in cells treated by surfactants at 24 h. In conclusion, Cremophor EL and RH40 in concentrations corresponding to clinical doses caused endothelial and epithelial toxicity. Endothelial cells were more sensitive to surfactant treatment than epithelial cells, and Cremophor EL was more toxic than RH40 in both cell types. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... When translating these values to molarity, Kolliphor RH40 was non-cytotoxic in human cells up to 0.3-0.6 mM, Poloxamer 188 up to 0.3-30 mM, and of Tween 80 up to 0.3higher than 2.4 mM. It is reported that emulsifiers with high HLB values are less cytotoxic than lipophilic emulsifiers with lower HLB value, since higher lipophilicity of a molecule can cause higher risk of perturbation in cell membranes (Kiss et al., 2013). This may explain the relatively low cytotoxicity of the selected emulsifiers. ...
... This may explain the relatively low cytotoxicity of the selected emulsifiers. The higher cytotoxicity of Tween 80 in the A549 cells may be related to the available unsaturated fatty acid esters of sorbitol (oleic acid and linoleic acid) in this molecule (Kiss et al., 2013). Cytotoxicity screenings in the murine macrophages DMBM-2, J774A.1 and RAW 264.7 showed variable results (Fig. 2). ...
... Kolliphor RH40 was tested in pure form with controversial results. On Caco-2 cells only concentrations of > 10 mg/ml caused adverse effects, while in endothelial cells already < 0.1 mg/ml acted cytotoxic (Kiss et al., 2013). Kolliphor RH40 containing micelles, on the other hand, did not cause cytotoxicity at the concentration of 0.100 mg/ml in RAW 264.7 macrophages (Shao et al., 2017). ...
Article
Introduction: The pulmonary route is a non-invasive administration route that receives growing attention. The challenge for formulation development of orally inhaled formulations is, however, the limited number of approved excipients. Lipid nanoparticles are desired drug delivery systems for inhalation because lipids are biocompatible. However, addition of emulsifiers to stabilize the formulation may cause toxic effects. Alveolar epithelial cells and alveolar macrophages are the main cell types that get in contact with inhaled formulations in the deep lung. The different cell types are supposed to differ in the extent of particle uptake. Kolliphor RH40, Poloxamer 188, and Tween 80 are approved for use in oral formulations and widely used in the academic field for manufacturing of lipid nanoparticles. However, little is known about their pulmonary toxicity. Methods: Cytotoxicity of Kolliphor RH40, Poloxamer 188, and Tween 80 was studied by integration into solid lipid nanoparticles loaded with itraconazole as model drug. Cytotoxicity of the formulations was assessed in human alveolar epithelial cells and human and murine macrophages and correlated to cell uptake. Results: The tested emulsifiers showed overall low cytotoxicity with less pronounced adverse effects in human cells than in murine macrophages. Cellular uptake of Poloxamer 188 containing lipid nanoparticles was decreased in macrophages, while uptake of lipid nanoparticles with the other emulsifiers was similar in epithelial cells and phagocytes. Conclusion: The tested emulsifiers appear suitable for use in pulmonary applications. Due to larger cell size and lower proliferation rate human cells showed lower cytotoxicity than the murine cells. Being human cells, they appear more suitable for the screening of adverse effects in human lungs.
... Cremophor EL is approved for clinical use and is a component of many marketed products, including Taxol ® and Sandimmune ® for I.V. infusion. Clinical application of Cremophor EL-based formulations has raised concerns related to many SAA-related adverse effects reported, mainly anaphylactoid hypersensitivity reactions [80][81][82]. However, the observed effects are associated with relatively high blood concentrations of Cremophore EL achieved by I.V. infusion of the therapeutic doses of respective drug products; besides, prolonged exposure time of up to 24 h is sometimes dictated by therapeutic protocols. ...
... As for in-vitro toxicity in cell monolayers, cytotoxic effects are known to be cell and formulation type dependent. The cell tolerable concentration limit of Cremophor EL was reported to be 5 mg/mL in the Caco-2 epithelial cell line down to 0.1 mg/mL in the brain endothelial cell line [81]. In A549 cells, cell tolerable concentration of Cremophor EL was calculated as 50 µg/mL, but importantly, the study tested for the toxicity of Cremophor EL in combination with ethanol at a 1:1 ratio, that is, the vehicle employed in IV Taxol ® injections [80]. ...
Article
Full-text available
Oral tadalafil (TD) proved promising in treating pediatric pulmonary arterial hypertension (PAH). However, to ensure higher efficacy and reduce the systemic side effects, targeted delivery to the lungs through nebulization was proposed as an alternative approach. This poorly soluble drug was previously dissolved in nanoemulsions (NEs). However, the formulations could not resist aqueous dilution, which precluded its dilution with saline for nebulization. Thus, the current study aimed to modify the previous systems into dilutable TD-NEs and assess their suitability for a pulmonary application. In this regard, screening of various excipients was conducted to optimize the former systems; different formulations were selected and characterized in terms of physicochemical properties, nebulization performance, stability following sterilization, and biocompatibility. Results showed that the optimal system comprised of Capmul-MCM-EP:Labrafac-lipophile (1:1) (w/w) as oil, Labrasol:Poloxamer-407 (2:1) (w/w) as surfactant mixture (Smix) and water. The optimum formulation P2TD resisted aqueous dilution, exhibited reasonable drug loading (2.45 mg/mL) and globule size (25.04 nm), acceptable pH and viscosity for pulmonary administration, and could be aerosolized using a jet nebulizer. Moreover, P2TD demonstrated stability following sterilization and a favorable safety profile confirmed by both in-vitro and in-vivo toxicity studies. These favorable findings make P2TD promising for the treatment of pediatric PAH.
... Though, Cremophor EL was shown to play a role in hypersensitivity reactions together with neurotoxicity, nephrotoxicity, and cardiotoxicity linked with i.v. delivery of paclitaxel (Gelderblom et al., 2001;Kiss et al., 2013;Pangeni et al., 2016). Transcutol HP was also employed in w/o part as a surfactant and w/o/w part as co-surfactant for the preparation of NE which also decreases interfacial tension and development of mechanical barriers to coalescence. ...
... It is very important to optimize the S mix ratio for the oral delivery of NE to enhance the entropy of dispersion and area of interfacial with decrement of interfacial-tension followed by free energy of the system for the thermodynamically stable system. It is also very important to play a role for oil-phase-to-Smixratio at the time of development of NE in which surfactants were also reduces interfacial-tension and decrement of globule-size (Gelderblom et al., 2001;Kiss et al., 2013;Pangeni et al., 2016). In this way, it reduces the globule-size from 153.23 to 51.64 nm with enhance in more surfactant come to closer at the site of adsorption & the construction of a extra closely-packedsurfactant film on the position of interface between to oil & water resulted a strongest stabilization. ...
Article
Full-text available
5-Fluorouracil (5-FU) is a drug of choice for colorectal-cancer. But oral therapeutic efficacy of 5-FU is restricted due to their very little bioavailability because of poor membrane permeability and GIT-absorption. We have developed a multiple nanoemulsion (w/o/w i.e. 5-FU-MNE) in which 5-FU incorporated to improve their oral-absorption. Globule-size of opt-5-FU-MNE was 51.64 ± 2.61 nm with PDI and ZP 0.101 ± 0.001 and −5.59 ± 0.94, respectively. In vitro 5-FU-release and ex vivo permeation studies exhibited 99.71% release and 83.64% of 5-FU from opt-nanoformulation. Cytotoxic in vitro studies-exhibited that 5-FU in opt-5-FU-MNE was 5-times more potent than 5-FU-S on human-colon-cancer-cell-lines (HT-29). The enhanced Cmax with AUC0-8h with opt-5-FU-MNE was shown extremely significant (p < 0.001) in wistar rat’s plasma in the comparison of oral and i.v. treated group of 5-FU-S by PK-observations. Furthermore, opt-5-FU-MNE was showed much more significant (p < 0.001) results as compared to 5-FU-S (free) on cell lines for human colon cancer (HT-29).
... The developed model was used to predict the size of additional formulations (F10 to F16): the obtained residuals (difference between the microemulsion size obtained experimentally Table 1). The optimal formulation was selected within the feasibility domain according to the following criteria: (i) the surfactant content had to be lower than 70% in order to minimise in vivo system toxicity [37,38], (ii) the EtOH content should not exceed the limit approved in medicinal products [39], and (iii) the oil content had to be high enough to emulsify the system but low enough to guarantee a high drug loading since the BI solubility in oil was very low. On these grounds, the SMEDDS F12, composed of 70% surfactant, 10% oil, 10% co-solvent Transcutol® HP, and 10% EtOH (Table 1; Fig. 2), was selected for further studies. ...
... Cell viability values higher than 70% were observed for blank SMEDDS c and S-SMEDDS I and their drug-loaded counterparts at system concentrations up to 1.3 mg·mL −1 . Such system concentration ensured that the Kolliphor® RH40 amount was below the reported limit of toxicity, since Kolliphor® RH40 in high amount was reported to damage Caco-2 cells by promoting oxidative stress and inhibition of the cardiac mitochondrial respiration [37]. Therefore, the ability of SMEDDS c and S-SMEDDS I to significantly reduce the drug toxicity on intestinal cells is a major advantage in the context of oral administration, as previously reported [46]. ...
Article
This study explored the design of supersaturable self-microemulsifying drug delivery systems (S-SMEDDS) to address poor solubility and oral bioavailability of a novel benzimidazole derivative anticancer drug (BI). Firstly, self-microemulsifying drug delivery systems SMEDDS made of Miglyol® 812, Kolliphor® RH40, Transcutol® HP, and ethanol were prepared and loaded with the BI drug. Upon dispersion, the systems formed neutrally charged droplets of around 20 nm. However, drug precipitation was observed following incubation with simulated gastric fluid (pH 1.2). Aiming at reducing this precipitation and enhancing drug payload, supersaturable systems were then prepared by adding 1% hydroxypropyl cellulose as precipitation inhibitor. Supersaturable systems maintained a higher amount of drug in a supersaturated state in gastric medium compared with conventional formulations and were stable in simulated intestinal medium (pH 6.8). In vitro cell studies using Caco-2 cell line showed that these formulations reduced in a transient manner the transepithelial electrical resistance of the monolayers without toxicity. Accordingly, confocal images revealed that the systems accumulated at tight junctions after a 2 h exposure. In vivo pharmacokinetic studies carried out following oral administration of BI-loaded S-SMEDDS, SMEDDS, and free drug to healthy mice showed that supersaturable systems promoted drug absorption compared with the other formulations. Overall, these data highlight the potential of using the supersaturable approach as an alternative to conventional SMEDDS for improving oral systemic absorption of lipophilic drugs.Graphical abstract
... Cremophor RH40 (polyoxyl 40 castor oil) is a colorless, tasteless and transparent non-ionic surfactant. It is a solubilizing pharmaceutic aid that is widely used in oral formulations, due to nontoxicity and stability over a wide temperature range [52]. Poloxamers are hydrophilic nonionic surfactants. ...
... They have high hydrophilic-lipophilic balance (HLB) values, which are 29 for Pluronic F-68 and 22 for Pluronic F-127. Since they are nonionized, this makes them less toxic and safe to use orally [52]. Avicel PH-102 is an ideal binder for the wet granulation process. ...
Article
Full-text available
Aim: To study the impact of various permeability enhancers on fexofenadine bioavailability. Furthermore, to predict the potential effect of Cremophor ® RH 40 on fexofenadine pharmacokinetics at higher doses using Biopharmaceutical Classification System criteria. Experimental methods: The effect of the dose increase (60–360 mg) on the dissolution and permeability behavior of fexofenadine-Cremophor RH 40 formulations was studied in humans. The Biopharmaceutical Classification System criteria of the drug was determined. Results & conclusion: Cremophor RH 40 improved the dissolution and bioavailability of fexofenadine. The pharmacokinetics increased linearly with the dose increase. Absorption number (A n ) was significantly increased after addition of Cremophor RH 40 in comparison to an unprocessed drug. Similar A n values were observed throughout the same dose range. The dose number (D 0 ) values were <1 whereas, all the dissolution number (D n ) values were >1 at the same dose level.
... Cremophor RH60 did not reduce the cell viability of the epithelial cells at either the 2.5 or 5 mg/mL concentration for 6 h (Figure 4). These results are in accordance with our previous studies, where concentrations of Cremophor RH40 less than 10 mg/mL were non-toxic for different cultured epithelial cells [25,26]. ...
... Cremophor RH60 did not reduce the cell viability of the epithelial cells at either the 2.5 or 5 mg/mL concentration for 6 h (Figure 4). These results are in accordance with our previous studies, where concentrations of Cremophor RH40 less than 10 mg/mL were non-toxic for different cultured epithelial cells [25,26]. Statistical analysis: One-way ANOVA followed by Dunnett's test. ...
Article
Full-text available
The aim of this study was to perform a preformulation study of dexamethasone (DXM)-loaded nanostructured lipid carriers (NLCs) for ocular use. Lipid screening was applied to find the most suitable solid and liquid lipids and surfactant for the NLC formulation. The visual observation was proved with XRD measurements for the establishment of the soluble state of DXM. Thermoanalytical measurements indicated that the most relevant depression of the crystallinity index could be ensured when using a 7:3 solid lipid:oil ratio. In order to optimize the NLC composition, a 2 3 full factorial experimental design was used. It was established that each independent factor (lipid, DXM, and surfactant concentration) had a significant effect on the particle size while in the case of entrapment efficiency, the DXM and surfactant concentrations were significant. Lower surfactant and lipid concentrations could be beneficial because the stability and the entrapment efficacy of NLCs were more favorable. The toxicity tests on human cornea cells indicated good ophthalmic tolerability of NLCs. The in vitro drug release study predicted a higher concentration of the solute DXM on the eye surface while the Raman mapping penetration study on the porcine cornea showed a high concentration of nanocarriers in the hydrophylic stroma layer.
... 40,41 Tween 80, a polyoxyethylene sorbitan monooleate (average HLB value of 15), has been deemed as a low-cost and powerful emulsifier. [42][43][44] Cremophor RH40, a polyoxyl 40 hydrogenated castor oil (HLB value ranging [14][15][16], is an efficient emulsifier used to formulate several drugs such as paclitaxel and teniposide. 44 Pluronic F68 (Poloxamer 188), a PEO-PPO-PEO-triblock copolymer (HLB value of 29), is known to enhance the drug solubility and formulation stability in drug delivery system. ...
... [42][43][44] Cremophor RH40, a polyoxyl 40 hydrogenated castor oil (HLB value ranging [14][15][16], is an efficient emulsifier used to formulate several drugs such as paclitaxel and teniposide. 44 Pluronic F68 (Poloxamer 188), a PEO-PPO-PEO-triblock copolymer (HLB value of 29), is known to enhance the drug solubility and formulation stability in drug delivery system. 45,46 Among the various selected surfactants, HS-15 was therefore selected as the surfactant in Abbreviations: BD-sNeDDs, BD-loaded self-nanoemulsifying drug delivery system; BD-suspension, BD suspended in 0.5% sodium carboxymethyl cellulose solution; Tlr4, toll-like receptor 4; MyD88, myeloid differentiation primary response 88; TraF-6, TNF receptor-associated factor 6; NF-κB, nuclear factor-kappa B; aZa, azathioprine; TNBs, trinitrobenzenesulfonic acid; BDl-sNeDDs, low-dosage BD-loaded self-nanoemulsifying drug delivery system; BDM-sNeDDs, medium-dosage BD-loaded selfnanoemulsifying drug delivery system; BDh-sNeDDs, high-dosage BD-loaded self-nanoemulsifying drug delivery system. ...
Article
Full-text available
Background: Bruceine D (BD) is a major bioactive component isolated from the traditional Chinese medicinal plant Brucea javanica which has been widely utilized to treat dysentery (also known as ulcerative colitis [UC]). Methods: To improve the water solubility and absolute bioavailability of BD, we developed a self-nanoemulsifying drug delivery system (SNEDDS) composing of MCT (oil), Solutol HS-15 (surfactant), propylene glycol (co-surfactant) and BD. The physicochemical properties and pharmacokinetics of BD-SNEDDS were characterized, and its anti-UC activity and potential mechanism were evaluated in TNBS-induced UC rat model. Results: The prepared nanoemulsion has multiple beneficial aspects including small mean droplet size, low polydispersity index (PDI), high zeta potential (ZP) and excellent stability. Transmission electron microscopy showed that nanoemulsion droplets contained uniform shape and size of globules. Pharmacokinetic studies demonstrated that BD-SNEDDS exhibited enhanced pharmacokinetic parameters as compared with BD-suspension. Moreover, BD-SNEDDS significantly restored the colon length and body weight, reduced disease activity index (DAI) and colon pathology, decreased histological scores, diminished oxidative stress, and suppressed TLR4, MyD88, TRAF6, NF-κB p65 protein expressions in TNBS-induced UC rat model. Conclusion: These results demonstrated that BD-SNEDDS exhibited highly improved oral bioavailability and advanced anti-UC efficacy. In conclusion, our current results provided a foundation for further research of BD-SNEDDS as a potential complementary therapeutic agent for UC treatment.
... Another huge advantage of this technique is that impedimetry is a real-time measurement which gives us the opportunity to characterize the dynamics of adhesion. A variety of applications using impedimetry has been developed such as measurement of cell proliferation, 9 viability, 10 cellular permeability, 11 and cytotoxicity 12,13 as well as a number of systems using impedancebased approach have been also developed. While impedimetry has been used in research for decades in a number of ways, no clinical application has been developed yet. ...
... The xCELLigence system is a real-time technique for measurement of cell adhesion using an impedimetric approach. Its reliability and high sensitivity have been demonstrated with a variety of cell types and applications.[9][10][11][12][13] Platelets are cell fragments with considerably smaller dimensions than whole cells; therefore, a highly sensitive method is needed for detection of changes in adhesion and spreading. ...
Article
Introduction: Thrombogenesis plays an important role in today's morbidity and mortality. Antithrombotics are among the most frequently prescribed drugs. Thorough knowledge of platelet function is needed for optimal clinical care. Platelet adhesion is a separate subprocess of platelet thrombus formation; still, no well-standardized technique for the isolated measurement of platelet adhesion exists. Impedimetry is one of the most reliable, state-of-art techniques to analyze cell adhesion, proliferation, viability, and cytotoxicity. We propose impedimetry as a feasible novel method for the isolated measurement of 2 significant platelet functions: adhesion and spreading. Methods: Laboratory reference platelet agonists (epinephrine, ADP, and collagen) were applied to characterize platelet functions by impedimetry using the xCELLigence SP system. Platelet samples were obtained from 20 healthy patients under no drug therapy. Standard laboratory parameters and clinical patient history were also analyzed. Results: Epinephrine and ADP increased platelet adhesion in a concentration-dependent manner, while collagen tended to have a negative effect. Serum sodium and calcium levels and age had a negative correlation with platelet adhesion induced by epinephrine and ADP, while increased immunoreactivity connected with allergic diseases was associated with increased platelet adhesion induced by epinephrine and ADP. ADP increased platelet spreading in a concentration-dependent manner. Conclusion: Impedimetry proved to be a useful and sensitive method for the qualitative and quantitated measurement of platelet adhesion, even differentiating between subgroups of a healthy population. This novel technique is offered as an important method in the further investigation of platelet function.
... Despite its outstanding antitumor properties, taxol is difficult to isolate, resulting in very low yields and high costs [104] The first formulation of taxol contains a cremophor EL (polyethoxylated castor oil), which functions to increase the solubility of taxol [105]. However, the cremophor was later found to be too toxic to living cells, including endothelial and epithelial cells [106]. Therefore, further research was conducted on taxols that lead to nanoparticle involvement. ...
Article
Full-text available
Phytochemicals or secondary metabolites are substances produced by plants that have been shown to have many biological activities, providing a scientific basis for using herbs in traditional medicine. In addition, the use of herbs is considered to be safe and more economical compared to synthetic medicine. However, herbal medicines have disadvantages, such as having low solubility, stability, and bioavailability. Some of them can undergo physical and chemical degradation , which reduces their pharmacological activity. In recent decades, nanotechnology-based herbal drug formulations have attracted attention due to their enhanced activity and potential for overcoming the problems associated with herbal medicine. Approaches using nanotechnology-based delivery systems that are biocompatible, biodegradable, and based on lipids, polymers, or nanoemulsions can increase the solubility, stability, bioavailability, and pharmacological activity of herbals. This review article aims to provide an overview of the latest advances in the development of nanotechnology-based herbal drug formulations for increased activity, as well as a summary of the challenges these delivery systems for herbal medicines face.
... Cremophor EL is a non-ionic surfactant with relatively low toxicity compared to other ionic surfactants; therefore, it is generally known to be safe and biocompatible. It has a high HLB value (HLB ~12) and exhibits high emulsifying properties; therefore, it is widely used in the oral formulation of microemulsions and SMEDDS [40]. Labrasol® can enhance the permeation of macromolecules into the gastrointestinal tract in a transient, reversible, and molecular weight-dependent manner [41]. ...
Article
To overcome the low oral bioavailability of insulin, we hypothesized that the insulin-hydrophobic ion pairing (HIP) complex incorporated self-microemulsifying drug delivery system (SMEDDS) would be beneficial. In the present study, an oral insulin delivery system was developed and estimated using the HIP technique and SMEDDS. Further insulin-HIP complexes were characterized using various spectroscopical techniques. Additionally, insulin-HIP complexes were subjected to analysis of complexes' conformational stability in the real physiological solution using computational approaches. On the other hand, in vitro, and in vivo studies were carried out to investigate the permeability and hypoglycemic effect. Subsequently, in an in vitro non-everted gut sac study, the apparent permeability coefficient (Papp) was approximately 8-fold higher in the colon than in the jejunum, and the HIP-incorporated SMEDDS showed an approximately 3-fold higher Papp value than the insulin solution. The hypoglycemic effect after in situ colon instillation, the HIP complex between insulin and sodium docusate-incorporated SMEDDS showed a pharmacological availability of 2.52 ± 0.33 % compared to the subcutaneously administered insulin solution. Thus, based on these outcomes, it can be concluded that the selection of appropriate counterions is important in developing HIP-incorporated SMEDDS, wherein this system shows promise as a tool for oral peptide delivery systems.
... Untargeted delivery results in a significantly less amount of drug accumulating at the target site [5,6]. Moreover, the toxic excipients being used in traditional therapy cause severe health issues in already weak cancer patients [7,8]. Paclitaxel (PTX) is a widely used drug in breast cancer chemotherapy. ...
Article
Full-text available
Delivering water-insoluble drugs, such as paclitaxel (PTX), to the tumor site is a challenge. Exosomes are capable of delivering “cargo” to the target site without causing adverse effects, unlike synthetic nanoparticles. Here, we have prepared PTX-loaded exosomes from breast cancer cells. This formulation is further explored for its activity against breast cancer cell lines. Our data indicate that the exosomes are more efficient than free paclitaxel and liposomal paclitaxel in inhibiting the cancer cell growth. The primary reason for this superior activity is determined to be exosomes’ ability to deliver more amount of drug in the cytosol. However, the exact mechanism by which exosomes deliver the drug across the cell membrane still needs to be explored.
... Herein, the combination of GSP and immune checkpoint blockade therapy was evaluated as a potential therapy. However, the vehicle of GSP was based on Cremophor RH 40 [31,41], which causes hypersensitivity reactions and toxicity [42]. ...
Article
Immunotherapy is blooming in recent years. However, this therapy needs to overcome off-target effects, cytokine release syndrome, and low responses in the ‘cold’ tumor environment. Herein, various combinations of immunotherapies and chemotherapies were proposed to transform ‘cold’ tumors into ‘hot’ tumors to enhance the efficacy of immunotherapies. In this study, we prepared a biocompatible ganetespib (GSP)-loaded PEGylated nanocarriers (NCs) with a thin-film method, which exhibited a small particle size (~220.6 nm), high drug loading (~5.8%), and good stability. We designed and produced the cluster of differentiation 3 (CD3)/programmed death ligand 1 (PD-L1)/methoxy-polyethylene glycol (mPEG) trispecific antibodies (TsAbs) as bispecific T-cell engagers (BiTEs) to non-covalently bind the GSP-NCs via anti-mPEG fragment and endowed the GSP-NCs with a targeting ability and immunotherapeutic potential to activate cytotoxic T cells. Decoration of the GSP-NCs with TsAbs (BiTEs-GSP-NCs) significantly promoted the cellular uptake and showed synergistic effects through respective anti-PD-L1 and anti-CD3 activation of T cell-mediated cytotoxicity. In vivo tumor-inhibition studies also showed that the BiTEs-GSP-NCs could inhibit tumor growth with the GSP chemodrug and increase T-cell infiltration. This study provides a promising drug delivery strategy for cancer immunochemotherapy.
... They form "ex tempore" small (10-30 nm) classic micelles besides physically coating the particles to keep them suspended or solubilized [5]. The concentrations of CR 40 in oral formulations ranges from 2 to 600 and from 25 to 405 mg (FDA database for Inactive Ingredients in Approved Drug Products) [6]. Soluplus ® (SP) is a novel solubilizing amphiphilic graft copolymer (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) offering better solubility enhancement in combination with rapid drug release due to spontaneously micelle formation (~80 nm) above the critical micelle concentration (CMC). ...
Article
Full-text available
In this study, the effect of Cremophor® RH 40 (CR 40) classic micelles and Soluplus® (SP) polymeric micelles were investigated on a novel granule-type drug-delivery system containing megestrolacetate (MGA). Using a risk assessment-based approach on the formulation via melt technology resulted in the formation of these granules, presented as the dosage, with proper particle size and flow characteristics. Due to the application of a eutectic carrier base composition, gentle process conditions were reached, retaining the crystalline structure of the carrier system and allowing for the proper distribution of MGA in the granules. The increased water solubility (0.111 mg/mL to 2.154 mg/mL), and the decreased nano particle size (102.27 nm) with uniform distribution (polydispersity index of 0.259) and colloid stability (zeta potential of −12.99 mV) resulted in SP polymeric micelles prevailing over CR 40 micelles in this gastric dissolution study, performed in biorelevant fasted and fed state drug-release media. Mathematical characterization and kinetic model fitting supported the fast drug-release mechanism of polymeric micelles over micelles. The value-added polymeric micelle-containing formulation developed can be successfully administered perorally and the enhanced drug release offers the possibility of greater drug absorption in the gastrointestinal tract
... In previous observations, the studied non-ionic surfactants incurred no significant cytotoxicity in micellar compositions at relatively high concentrations [25]. However, a significant increase in the concentration of these surfactants in culture medium was reported to reduce cell viability [26,27]. Furthermore, the cationic properties of polymers, commonly associated with the cell membrane and cellular metabolic disturbances, did not show cytotoxic potential [28]. ...
Article
Full-text available
Context: Overcoming the intestinal mucosal barrier can be a challenge in drug delivery. Nanoemulsions with negative zeta potentials can effectively permeate the mucus layer, but those with positive zeta potentials are better taken up by cells; a nanoemulsion with capricious zeta potential from negative to positive can achieve both good permeation and high uptake. Objective: This study aimed to develop dual-acting zeta-potential-amphoteric micelles enabling optimal muco-permeation and enhancement of cellular uptake. Methods: A micellar pre-concentrate was prepared from 15% Labrasol, 15% Kolliphor EL, 30% Kolliphor RH 40, and 40% dimethylsulfoxide. The micellar pre-concentrate was loaded with anionic stearic acid (SA), forming ionic complexes with cationic polymers at a ratio of 25:1 with Eudragit RS 100 and Eudragit RL 100. Blank micelles and those containing complexes were separately diluted in physiological buffers and examined for their droplet sizes, polydispersity indices (PDIs), zeta potentials, and cytotoxicity. The SA release from the micellar complexes was evaluated in 0.1 mM phosphate buffer (pH 6.8) containing 0.001% fluorescein, thereby enabling an instant decrease in fluorescence. Finally, the micelles were loaded with the model drug fluorescein diacetate (FDA) and evaluated for their muco-permeation behavior and cellular uptake. Results: The micellar dilutions formed micelles at the critical micelle concentration (CMC) of 312 µg/mL and showed a uniform average droplet size of 14.2 nm, with a PDI < 0.1. Micellar dilutions were non-cytotoxic when used at 1:100 in a physiological medium. Micelles loaded with ionic complexes achieved a sustained release of 95.5 ± 3.7% of the SA in 180 min. Moreover, the zeta potential of the complex-loaded micelles shifted from -5.4 to +1.8 mV, whereas the blank micelles showed a stabilized zeta potential of -10 mV. Furthermore, the negatively charged blank and complex-loaded micelles exhibited comparable muco-permeation, with an overall average of 58.2 ± 3.7% diffusion of FDA. The complex-loaded micellar droplets, however, provided a significantly higher cellular uptake of the model drug FDA (2.2-fold, p ≤ 0.01) Conclusion: Due to undergoing a shift in zeta potential, the modified micelles significantly enhanced cellular uptake while preserving mucus-permeating properties.
... We have successfully tested the cellular effects of pharmaceutical excipients and peptides by impedance kinetics in our previous studies. [32][33][34] For background measurements 50 μL of cell culture medium was added to the wells, then cells were seeded at a density of 5×10 3 cells/well to 96-well plate with gold electrodes (E-plate 96, ACEA Biosciences) coated with collagen. Cells were cultured for 4-5 days in a CO 2 incubator at 37°C and monitored every 10 minutes until the end of experiments. ...
Article
Full-text available
Background: Optimal transcorneal penetration is necessary for ocular therapy; meanwhile, it is limited by the complex structure and defensive mechanisms of the eye. Antimicrobial stability of topical ophthalmic formulations is especially important. According to previous studies, the mostly used preservative, benzalkonium-chloride is irritative and toxic on corneal epithelial cells; therefore, novel non-toxic, antimicrobial agents are required. In this study, prednisolone-containing ophthalmic formulations were developed with expected optimal permeation without toxic or irritative effects. Methods: The toxicity and permeability of prednisolone-containing eye drops were studied on a human corneal epithelial cell line (HCE-T) and ex vivo cornea model. The lipophilic drug is dissolved by the formation of cyclodextrin inclusion complex. Zinc-containing mucoadhesive biopolymer was applied as an alternative preservative agent, whose toxicity was compared with benzalkonium-chloride. Results: As the results show, benzalkonium-chloride-containing samples were toxic on HCE-T cells. The biopolymer caused no cell damage after the treatment. This was confirmed by immunohistochemistry assay. The in vitro permeability was significantly higher in formulations with prednisolone-cyclodextrin complex compared with suspension formulation. According to the ex vivo permeability study, the biopolymer-containing samples had significantly lower permeability. Conclusion: Considering the mucoadhesive attribute of target formulations, prolonged absorption is expected after application with less frequent administration. It can be stated that the compositions are innovative approaches as novel non-toxic ophthalmic formulations with optimal drug permeability.
... DXM exerted a cell layer tightening effect that is visible in all NLC treatment groups as an increased level of impedance [26,27]. We found good correlation of cell impedance data with conventional cellular toxicity assays, like MTT and lactate dehydrogenase release tests and cellular morphology in a previous study [28]. ...
Article
Full-text available
Generally, topically applied eye drops have low bioavailability due to short residence time and low penetration of the drug. The aim of the present study was to incorporate dexamethasone (DXM) into nano lipid carriers (NLC), which contain mucoadhesive polymer, in order to increase the bioavailability of the drug. A 23 factorial experimental design was applied, in which the three factors were the polymer, the DXM, and the emulsifier concentrations. The samples were analyzed for particle size, zeta potential, polydispersity index, and Span value. The significant factors were identified. The biocompatibility of the formulations was evaluated with human corneal toxicity tests and immunoassay analysis. The possible increase in bioavailability was analyzed by means of mucoadhesivity, in vitro drug diffusion, and different penetration tests, such as in vitro cornea PAMPA model, human corneal cell penetration, and ex vivo porcine corneal penetration using Raman mapping. The results indicated that DXM can be incorporated in stable mucoadhesive NLC systems, which are non-toxic and do not have any harmful effect on cell junctions. Mucoadhesive NLCs can create a depot on the surface of the cornea, which can predict improved bioavailability.
... Kinetics of cellular responses to niosome treatments were monitored by impedance measurement (RTCA-SP; ACEA Biosciences, San Diego, CA, USA), which is label-free, real-time, non-invasive test and correlates linearly with viability of cells [41]. Furthermore, this method correlates well with other conventional cell viability assays, as shown in our previous article [42]. After background measurements with culture medium, cells were seeded at a density of 6 × 10 3 cells/well in 96-well plates with integrated gold electrodes (E-plate 96, ACEA Biosciences, San Diego, CA, USA). ...
Article
Full-text available
Inefficient drug delivery across the blood–brain barrier (BBB) and into target cells in the brain hinders the treatment of neurological diseases. One strategy to increase the brain penetration of drugs is to use vesicular nanoparticles functionalized with multiple ligands of BBB transporters as vehicles. Once within the brain, however, drugs must also be able to reach their therapeutic targets in the different cell types. It is, therefore, favorable if such nanocarriers are designed that can deliver their cargo not only to brain endothelial cells, but to other cell types as well. Here, we show that alanine-glutathione dual-targeting of niosomes enhances the delivery of a large protein cargo into cultured cells of the neurovascular unit, namely brain endothelial cells, pericytes, astrocytes and neurons. Furthermore, using metabolic and endocytic inhibitors, we show that the cellular uptake of niosomes is energy-dependent and is partially mediated by endocytosis. Finally, we demonstate the ability of our targeted nanovesicles to deliver their cargo into astroglial cells after crossing the BBB in vitro. These data indicate that dual-labeling of nanoparticles with alanine and glutathione can potentially be exploited to deliver drugs, even biopharmacons, across the BBB and into multiple cell types in the brain.
... This method has been established to follow not only cell attachment and growth, but cell viability as well. We have verified by endpoint colorimetric tests and morphological methods that impedance changes can sensitively detect cell damage [23,29,33] and protection [23,29]. The RTCA-SP system (ACEA Biosciences, CA, USA) registers the impedance of cells automatically every 10 min. ...
Article
Full-text available
Background: Excitotoxicity is a central pathological pathway in many neurological diseases with blood-brain barrier (BBB) dysfunction. Kainate, an exogenous excitotoxin, induces epilepsy and BBB damage in animal models, but the direct effect of kainate on brain endothelial cells has not been studied in detail. Our aim was to examine the direct effects of kainate on cultured cells of the BBB and to test three anti-inflammatory and antioxidant drugs used in clinical practice, simvastatin, edaravone and dexamethasone, to protect against kainate-induced changes. Methods: Primary rat brain endothelial cell, pericyte and astroglia cultures were used to study cell viability by impedance measurement. BBB permeability was measured on a model made from the co-culture of the three cell types. The production of nitrogen monoxide and reactive oxygen species was followed by fluorescent probes. The mRNA expression of kainate receptors and nitric oxide synthases were studied by PCR. Results: Kainate damaged brain endothelial cells and made the immunostaining of junctional proteins claudin-5 and zonula occludens-1 discontinuous at the cell border indicating the opening of the barrier. The permeability of the BBB model for marker molecules fluorescein and albumin and the production of nitric oxide in brain endothelial cells were increased by kainate. Simvastatin, edaravone and dexamethasone protected against the reduced cell viability, increased permeability and the morphological changes in cellular junctions caused by kainate. Dexamethasone attenuated the elevated nitric oxide production and decreased the inducible nitric oxide synthase (NOS2/iNOS) mRNA expression increased by kainate treatment. Conclusion: Kainate directly damaged cultured brain endothelial cells. Simvastatin, edaravone and dexamethasone protected the BBB model against kainate-induced changes. Our results confirmed the potential clinical usefulness of these drugs to attenuate BBB damage.
... Impedance was measured at 10 kHz by an RTCA SP instrument (RTCA-SP instrument, ACEA Biosciences, San Diego, CA, USA). We have successfully tested the cellular effects of peptides and pharmaceutical excipients by impedance kinetics [23,27,28]. For background measurements, 50 µL cell culture medium was added to the wells. ...
Article
Full-text available
The potential of poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) to overcome the intestinal barrier that limits oral liraglutide delivery was evaluated. Liraglutide-loaded PLGA NPs were prepared by the double emulsion solvent evaporation method. In vitro release kinetics and enzymatic degradation studies were conducted, mimicking the gastrointestinal environment. The permeability of liraglutide solution, liraglutide-loaded PLGA NPs, and liraglutide in the presence of the absorption enhancer PN159 peptide was tested on the Caco-2 cell model. Liraglutide release from PLGA NPs showed a biphasic release pattern with a burst effect of less than 15%. The PLGA nanosystem protected the encapsulated liraglutide from the conditions simulating the gastric environment. The permeability of liraglutide encapsulated in PLGA NPs was 1.5-fold higher (24 × 10 −6 cm/s) across Caco-2 cells as compared to liraglutide solution. PLGA NPs were as effective at elevating liraglutide penetration as the tight junction-opening PN159 peptide. No morphological changes were seen in the intercellular junctions of Caco-2 cells after treatment with liraglutide-PLGA NPs, confirming the lack of a paracellular component in the transport mechanism. PLGA NPs, by protecting liraglutide from enzyme degradation and enhancing its permeability across intestinal epithelium, hold great potential as carriers for oral GLP-1 analog delivery.
... Los efectos adversos de algunos excipientes comúnmente usados en las formulaciones lipídicas han promovido la investigación de alternativas para mejorar la biodisponibilidad de los fármacos alterando lo menos posible la integridad del organismo [14,115]. Es así como los ácidos biliares PEGilados (BA-PEG, por sus siglas en inglés) se perfilan como una opción interesante para los tensioactivos tradicionales. ...
Article
Full-text available
Los grandes avances tecnológicos en la industria farmacéutica, que involucran el uso de la química combinatoria y el cribado de alto rendimiento, han conllevado al descubrimiento de muchas entidades químicas candidatas a fármacos que presentan baja solubilidad acuosa, debido a su elevada complejidad molecular, lo que hace difícil el desarrollo de productos con estas sustancias. Los sistemas de entrega de fármacos autoemulsificables (SEDDS) han generado un interés para el desarrollo farmacéutico porque son una alternativa efectiva para mejorar la biodisponibilidad de fármacos poco solubles en agua. Para describir el estado de conocimiento sobre estos sistemas se realizó una revisión sistemática en diferentes bases de datos sobre la literatura relacionada con los SEDDS a nivel nacional e internacional, logrando así describir los aspectos más relevantes sobre los SEDDS (tipos, composición, mecanismos para aumentar biodisponibilidad, caracterización, formulaciones). A pesar de las numerosas investigaciones realizadas durante los últimos años que muestran el potencial de los SEDDS para mejorar la biodisponibilidad de los fármacos poco solubles en agua, se pudo evidenciar que solo algunas sustancias activas han sido incluidas en estos sistemas y comercializadas exitosamente, esto debido a algunas limitaciones que indican la necesidad de un mayor entendimiento sobre estos sistemas.
... UV-vis spectra of these compounds were further recorded in physiological saline solution (PSS) and in PSS formulated with 1% Cremophor EL as a nonionic surfactant, which is widely used to improve the dissolution and delivery of drugs, such as Neoral® (Novartis, East Hanover, NJ), to evaluate the aggregation behavior in biologic environment [45]. As shown in Fig. 2, the Q-band of 1 in PSS greatly reduces in intensity with a red-shift of 24 nm in contrast to that in DMF (from 686 nm in DMF to 710 nm in PSS). ...
Article
A novel silicon(IV) phthalocyanine (SiPc) axially di-substituted with Arg-Arg-Arg oligopeptides (compound 4) has been synthesized and characterized. Meanwhile, two analogues have been used as controls. The effect of arginine number on the spectral properties, photosensitizing activities, partition coefficient, cellular uptake, and photodynamic antimicrobial activities have been investigated. The novel SiPc 4 containing tri-arginine oligopeptides exhibits an extremely high in vitro photodynamic activity not only against Gram-positive/negative bacteria but also against fungus, which is obviously superior to its analogues containing mono-arginine (compound 2) and di-arginine oligopeptides (compound 3). In addition, 4 shows a higher photodynamic inactivation against S.aureus, E.coli and C. albicans cells than methylene blue (MB), a known antimicrobial photosensitizer. The IC90 values against C. albicans for 4 is ca. 179-fold lower than that for MB. Moreover, the potent photodynamic effect of 4 has been further confirmed by in vivo animal model. These results indicate that the tri-arginine-modified SiPc is a highly promising photosensitizer for photodynamic antimicrobial therapy.
... The current dose applied in our study was 5.15 mg/mL (0.5%, 0.039 M) with an effective concentration lower than 0.1% (see below). In epithelial cells, concentrations of 5 mg/mL and above were toxic at incubation times relevant to the present study, while in endothelial cell culture cremophor had negative effects at concentrations higher than 0.1 mg/mL (Kiss et al., 2013). The threshold in the current study seems to be similar, as we observed significant effects on the potassium-induced contractility at concentrations that were at 2.56 mg/mL (0.25%, 0.019 M) or higher (Figure 3(b)). ...
Article
Full-text available
Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K⁺ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.
... As shown in Fig 2A and 2D, the O/W microemulsion region with Cremophor RH40 (HLB = 14-16) is larger than that with Cremophor EL (HLB = 12-14), because the larger the HLB value of the emulsifier is, the stronger the emulsifying capacity is. Moreover, the toxicity of Cremophor RH40 is smaller [27]. Therefore, Cremophor RH40 was selected as the emulsifier. ...
Article
Full-text available
The purpose of this study was to improve the solubility of resveratrol (Res) by a self-microemulsifying drug-delivery system (SMEDDS). Through a solubility experiment, the pseudoternary phase diagram and ternary phase diagram were used to optimize the Res SMEDDS formula. The optimum formulation consisted of 5% IPM, 20% PEG400, and 65% Cremophor RH40. The water solubility, stability, in vitro release and antioxidant activity of the Res SMEDDS were characterized. The Res solubility in the SMEDDS was at least 1,000 times compared to that in water. The average droplet size of the microemulsion was 28.00±1.67 nm and uniform distribution. The Res SMEDDS should be stored at low temperature and in the dark to avoid light conditions. Res SMEDDS was able to improve the in vitro release rate of Res, and the in vitro release of Res from Res SMEDDS was significantly faster that of Res powder and unaffected by pH value of media. Antioxidant assays showed that antioxidant activities of Res in Res SMEDDS were unaffected compared to Res powder. Cytotoxicity study indicated that Res SMEDDS at the concentration of less than 100 μM was safe. These results demonstrated the potential use of Res SMEDDS for oral administration of Res.
... Therefore, it is difficult to compare our data with data from the literature. In addition to the cited inhibition effect, the surfactants may also cause profound effects on cells, disturbing plasma membranes or removing their lipids (Hugger et al. 2002;Kiss et al. 2013). Apparently, the concentration of Cremophor RH40 present in the NLC was much lower than its CMC (critical micellar concentration) value (0.01% m/v, Matsaridou et al. 2012), which may have led to the effect of inhibition of Pgp efflux transporters. ...
Article
Full-text available
Hydrophobic bioactives can be more easily incorporated into food and have their bioavailability enhanced if nanostructured lipid carriers (NLC) are used as carriers. In the present study, beta-carotene-loaded NLC were produced by low emulsificationusing murumuru butter and a mixture of Span 80 and Cremophor RH40 as surfactants. Their average diameter was 35 nm andalpha-tocopherol was required to protect the encapsulated β-carotene. Besides the evaluation of their physicochemical stability, NLC were submitted to dynamic in vitrodigestion and cell viability assays with Caco-2 and HEPG cells. The bioaccessibility of beta-carotene in the dynamic system was about 42%.Regarding cell viability, results indicated NLC were toxic to the cell cultures tested. Such high toxicity is probably related to the type of surfactant used and tothe extremely reduced particle size, which may have led to an intense and fast permeation of the NLC through the cells.
... As a simple medium, it always supplemented with additives, or a high concentration of serum makes them suitable to grow mammalian cells Arora 2013). This medium is used to culture embryonic nerve cells, glial cells, and cell lines like SH-SY5Y cells, hBMECs, hCMECs, hNPCs, PC12 and SK-N-MC cells (Waheed Roomi et al. 2013;Aldick et al. 2007;Kiss et al. 2013;Ravichandran et al. 2011;Zheng et al. 2016). ...
Article
Full-text available
Globally, the central nervous system (CNS) disorders appear as the most critical pathological threat with no proper cure. Alzheimer’s disease (AD) is one such condition frequently observed with the aged population and sometimes in youth too. Most of the research utilizes different animal models for in vivo study of AD pathophysiology and to investigate the potency of the newly developed therapy. These in vivo models undoubtably provide a powerful investigation tool to study human brain. Although, it sometime fails to mimic the exact environment and responses as the human brain owing to the distinctive genetic and anatomical features of human and rodent brain. In such condition, the in vitro cell model derived from patient specific cell or human cell lines can recapitulate the human brain environment. In addition, the frequent use of animals in research increases the cost of study and creates various ethical issues. Instead, the use of in vitro cellular models along with animal models can enhance the translational values of in vivo models and represent a better and effective mean to investigate the potency of therapeutics. This strategy also limits the excessive use of laboratory animal during the drug development process. Generally, the in vitro cell lines are cultured from AD rat brain endothelial cells, the rodent models, human astrocytes, human brain capillary endothelial cells, patient derived iPSCs (induced pluripotent stem cells) and also from the non-neuronal cells. During the literature review process, we observed that there are very few reviews available which describe the significance and characteristics of in vitro cell lines, for AD investigation. Thus, in the present review article, we have compiled the various in vitro cell lines used in AD investigation including HBMEC, BCECs, SHSY-5Y, hCMEC/D3, PC-2 cell line, bEND3 cells, HEK293, hNPCs, RBE4 cells, SK-N-MC, BMVECs, CALU-3, 7W CHO, iPSCs and cerebral organoids cell lines and different types of culture media such as SCM, EMEM, DMEM/F12, RPMI, EBM and 3D-cell culture. Graphical Abstract Open image in new window
... Regarding medicines approval, it is not possible to justify the use of PEs that act to improve flux via transcellular perturbation by citing the side effects of drugs, excipients or food additives. However, a PE may not be listed as such in any given formulation, and therefore their use may be more comparable to other additives that have been shown to cause membrane perturbation in vitro (e.g., surfactants [119]). Comparison of PEs to established excipients does help to show that mild mucosal perturbation may be common. ...
Article
Full-text available
The application of permeation enhancers (PEs) to improve transport of poorly absorbed active pharmaceutical ingredients across the intestinal epithelium is a widely tested approach. Several hundred compounds have been shown to alter the epithelial barrier, and although the research emphasis has broadened to encompass a role for nanoparticle approaches, PEs represent a key constituent of conventional oral formulations that have progressed to clinical testing. In this review, we highlight promising PEs in early development, summarize the current state of the art, and highlight challenges to the translation of PE-based delivery systems into safe and effective oral dosage forms for patients.
... The effects of the treatments were followed for 48 h (N = 4-8). Impedance measurement is label-free, real time, noninvasive, and correlates linearly with adherence, growth, number and viability of cells [43]. The cell index at each time point was defined as (R n − R b )/15, where R n is the cell-electrode impedance of the well when it contains cells and R b is the background impedance of the well with the medium alone. ...
Article
Full-text available
Alzheimer’s disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid β (Aβ) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aβ oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural β-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aβ oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aβ, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aβ toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aβ binding.
... It is a non-ionic surfactant similar to Cremophor EL (PEG-35 Hydrogenated Castor Oil), a major component of Sandimmune ® injection. They both were reported to be toxic (37). Additionally, there are several reports showing that Cremophor EL influences the pharmacokinetics of various drugs including CyA (35,(38)(39)(40). ...
Article
Full-text available
Purpose: The aim of this study was to assess the pharmacokinetics of methoxy poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) micellar formulation of cyclosporine A (CyA) following oral administration in rats making comparisons with its commercial microemulsion formulation, Neoral®. Methods: PEO-b-PCL copolymer was synthesized and used to form micelles encapsulating CyA. The release of CyA from Neoral® and PEO-b-PCL as well as PEO-b-PCL degradation were assessed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Polymeric micellar CyA and Neoral® were administered by oral gavage to healthy Wistar rats. At predetermined intervals, rats (n=5 for each time point) were euthanized, samples of blood and plasma were collected and analyzed for CyA using an LC-MS/MS assay. Blood and plasma pharmacokinetic parameters of CyA in its polymeric micellar formulation were compared to those of Neoral®. Results: Polymeric micelles of CyA showed < 15 and 10% increase in diameter in SGF and SIF, respectively, within 24 h. PEO-b-PCL showed signs of minimal degradation when incubated for > 8 h in SGF, but was stable in SIF. Drug release in both SGF and SIF was comparable between the two formulations except for significantly higher release of CyA in SIF only at 24 h time point from Neoral®. Following oral administration (10 mg/kg), the blood AUC0-∞ and tmax of CyA in the polymeric micellar formulation was comparable to that for Neoral®. However, the Cmax of CyA-loaded PEO-b-PCL micelles was significantly (p < 0.05) higher than that obtained with Neoral® (2.10 ± 0.41 versus 1.40 ± 0.25 µg/mL, respectively). CyA had higher blood-to-plasma concentration ratios in polymeric micelles compared to Neoral®, in vivo. Conclusion: Our results show that PEO-b-PCL micelles can serve as stable and good solubilizing carriers for oral delivery of CyA providing similar pharmacokinetic profile to that of Neoral®.
... Cell index was defined as R n eR b at each time point of measurement, where R n is the cell-electrode impedance of the well when it contains cells and R b is the background impedance of the well with the medium alone. Cell index values reflect cell number and viability (Kiss et al., 2013;Bocsik et al., 2016). Expression of genes encoding solute carriers for glucose and alanine in isolated rat brain microvessels and in primary rat brain endothelial cells in co-culture with rat pericytes and astrocytes. ...
Article
Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles.
... We have successfully tested the cellular effects of peptides and pharmaceutical excipients by impedance kinetics. [28][29][30] For background measurements, a 50 µL cell culture medium was added to the wells; then, cells were seeded at a density of 6×10 3 cells/well to 96-well plate with gold electrodes (E-plate 96, ACEA Biosciences) coated with collagen. Cells were cultured for 5 days in a CO 2 incubator at 37°C and monitored every 10 min until the end of experiments. ...
Article
Full-text available
Purpose The article reports a wet milling process, where the planetary ball mill was combined with pearl milling technology to reach nanosize range of meloxicam (Mel; 100–500 nm). The main purpose was to increase the dissolution rate and extent of a poorly water-soluble Mel as nonsteroidal anti-inflammatory drug as well as to study its permeability across cultured intestinal epithelial cell layers. Methods Viscosity of milled dispersion and particle size distribution and zeta potential of Mel were investigated and differential scanning calorimeter and X-ray powder diffractometer were used to analyse the structure of the suspended Mel. Finally in vitro dissolution test and in vitro cell culture studies were made. Results It was found that the ratio of predispersion and pearls 1:1 (w/w) resulted in the most effective grinding system (200-fold particle size reduction in one step) with optimized process parameters, 437 rpm and 43 min. Nanosuspension (1% Mel and 0.5% poly[vinyl alcohol]) as an intermediate product showed a stable system with 2 weeks of holding time. This optimized nanosuspension enhanced the penetration of Mel across cultured intestinal epithelial cell layers without toxic effects. Conclusion The dissolution rate of Mel from the poly(vinyl alcohol) stabilized nanosuspension justified its applicability in the design of innovative per oral dosage form (capsule) in order to ensure/give a rapid analgesia.
... In human CMEC/D3 brain endothelial cells 0.001%, and in Caco-2, epithelial cells 0.5% were toxic, especially at longer incubations. 45 Others reported on acute toxicity of the heart and thymocytes and renal cells, [46][47][48] and axonal degeneration and demyelination of neurons. 49,50 These reports correspond well with our findings, showing a strong cytotoxic effect on preadipocytes at concentrations !0.07%. ...
Article
Full-text available
Freeze-drying, also known as lyophilization, significantly improves the storage, stability, shelf life, and clinical translation of biopharmaceuticals. On the downside, this process faces complex challenges, i.e., the presence of freezing and drying stresses for the active compounds, the uniformity and consistency of the final products, and the efficiency and safety of the reconstituted lyophilized formulations. All these requirements can be addressed by adding specific excipients that can protect and stabilize the active ingredient during lyophilization, assisting in the formation of solid structures without interfering with the biological and/or pharmaceutical action of the reconstituted products. However, these excipients, generally considered safe and inert, could play an active role in the formulation interacting with the biological cellular machinery and promoting toxicity. Any side effects should be carefully identified and characterized to better tune any treatments in terms of concentrations and administration times. In this work, various concentrations in the range of 1 to 100 mg/mL of cellobiose, lactose, sucrose, trehalose, isoleucine, glycine, methionine, dextran, mannitol, and (2-hydroxypropyl)-β-cyclodextrin were evaluated in terms of their ability to create uniform and solid lyophilized structures. The freeze-dried products were then reconstituted in the appropriate cell culture media to assess their in vitro cytotoxicity on both a healthy cell line (B-lymphocytes) and their tumoral lymphoid counterpart (Daudi). Results showed that at 10 mg/mL, all the excipients demonstrated suitable lyophilized solid structures and high tolerability by both cell lines, while dextran was the only excipient well-tolerated also up to 100 mg/mL. An interesting result was shown for methionine, which even at 10 mg/mL, selectively affected the viability of the cancerous cell line only, opening future perspectives for antitumoral applications.
Article
Oral delivery is considered the preferred route of administration due to its convenience and favorable compliance. However, this delivery often faces difficulties, such as poor solubility, limited absorption, and undesirable stability, especially for some volatile oils. The aim of this study was to develop self-emulsifying drug delivery systems (SEDDS) containing cinnamaldehyde (CA) to overcome these shortcomings. The CA-SEDDS were spherical and smooth with an average size of 14.96 ± 0.18 nm. Differential scanning calorimetry (DSC) and attenuated total reflection by Fourier transform infrared (ATR-FTIR) showed that CA has been successfully loaded into SEDDS. The accumulative release of CA-SEDDS (73.39%) was approximately 2.14-fold that of free CA when using simulated intestinal fluid as the release medium. A scanning electron microscope was used to observe the mucus network structure. Rheological tests found that CA-SEDDS can appropriately enhance the viscosity of the mucus system. We found from tissue distribution studies that CA was more widely distributed in various tissues in the CA-SEDDS group compared to the free CA group. The cinnamaldehyde and cinnamon acid also accumulated more in various tissues in the CA-SEDDS group than in the free CA group, especially in the kidney. These findings hinted that SEDDS exhibited lower irritation, good release, and penetration, which demonstrated great potential for utilizing CA. Our research supports the rational implications of SEDDS in delivering similar volatile substances by improving the solubility, mucus penetration, and stability, resulting in excellent clinical efficacy.
Article
Numerous preclinical studies provide evidence that curcumin, a polyphenolic phytochemical extracted from Curcuma longa (turmeric) has neuroprotective, anti-inflammatory and antioxidant properties against various neurological disorders. Curcumin neuroprotective effects have been reported in different animal models of epilepsy, but its potential effect attenuating brain glucose hypometabolism, considered as an early marker of epileptogenesis that occurs during the silent period following status epilepticus (SE), still has not been addressed. To this end, we used the lithium-pilocarpine rat model to induce SE. Curcumin was administered orally (300 mg/kg/day, for 17 days). Brain glucose metabolism was evaluated in vivo by 2-deoxy-2-[18F]Fluoro-D-Glucose ([18F]FDG) positron emission tomography (PET). In addition, hippocampal integrity, neurodegeneration, microglia-mediated neuroinflammation and reactive astrogliosis were evaluated as markers of brain damage. SE resulted in brain glucose hypometabolism accompanied by body weight (BW) loss, hippocampal neuronal damage and neuroinflammation. Curcumin did not reduce the latency time to the SE onset, nor the mortality rate associated to SE. Nevertheless, it reduced the number of seizures, and in the surviving rats, curcumin protected BW and attenuated the short-term glucose brain hypometabolism as well as the signs of neuronal damage and neuroinflammation induced by the SE. Overall, our results support the potential adaptogen-like effects of curcumin attenuating key features of SE-induced brain damage.
Article
Type 2 Diabetes Mellitus (T2DM) is one of the prevalent metabolic disorders and is the leading cause of death across the globe. In the present invention, we reported significant synergistic anti-diabetic efficacy of novel SGLT II inhibitor, Canagliflozin (CFZ) with natural phytoconstituent, Trigonelline (TGL) in fenugreek oil using a self-microemulsifying based formulation. The optimized liquid SMEDD formulation (NADF) containing fenugreek oil (18.11% w/w), vitamin E TPGS (53.40% w/w) and linalool (10.12% w/w) were optimized by Design of Experiments and produces nanoparticulate stable microemulsion in vitro. Conversion to solid SMEDDS (NADF-SD) was facilitated by adsorption onto natural biosorbent coffee husk. The optimized solid formulation containing CFZ was in amorphous state with enhanced surface area, negligible chemical incompatibility and uniform drug content. Scanning electron microscopy validated the powdered SMEDDS′ spherical form. The release behavior of CFZ in NADF-SD in diverse biorelevant environments is enhanced by in vitro dissolution. Furthermore, neither pure CFZ nor the marketed product had any food impact. The permeability of the jejunum intestinal segment in rats was remarkable, as validated by confocal microscopy. In comparison to a pure drug and a commercial product, these trials show a 3.99 and 2.56 fold increase in Cmax and a 2.23 and 1.56 fold increase in AUC0-24h, respectively. Through biochemical levels, urine glucose excretion investigations, and SGLT II expression in rats, pharmacodynamic examination of improved NADF-SD demonstrates enhanced anti-diabetic effectiveness of CFZ. In a nutshell, this natural lipid-based micro particulate system is well-suited for this drug's anti-diabetic activity and synergy with CFZ.
Article
In this study, a new self-microemulsifying drug delivery system (SMEDDS) based on pine essential oil has been designed and successfully developed to load cyclosporine A (CsA), a poorly water-soluble immunosuppressant, for oral CsA softgel manufacturing. SMEDDS were prepared from pine essential oil and Capmul MCM as oily phases, Kolliphor RH 40 as surfactant, polyethylen glycol 400 as co-surfactant/co-solvent and none volatized co-solvent utilizing the pseudo-ternary phase diagrams. CsA SMEDDS loading 9.09% of CsA, dispersed in aqueous environments (water, pH 1.2, 4.5 and 6.8 buffers), presented an average droplet size of about 160 nm with polydispersity index (PdI) below 0.25, and were stable after 6 h of self-emulsification. Cytotoxicity of CsA SMEDDS on LLC-PK1 cells demonstrated that CsA SMEDDS had an IC50 value greater than that of the reference product, suggesting product safety against nephrotoxicity. CsA SMEDDS was filled into oval-5 soft gelatin capsules (softgels) containing 0.5% glycine which could avoid the cross-linking process. Additionally, CsA SMEDDS softgels presented the in vitro release profiles equivalent to that of Sandimmun Neoral® at pH 1.2, 4.5 and 6.8. Therefore, the pine essential oil based SMEDDS was considered to be potential and suitable for developing softgels to enhance the oral bioavailability of CsA.
Article
In evaluating vitamin E (VE) nutritional status of preterm infants, it is essential that any data should be compared with those of healthy term infants, and never with those of adults. Moreover, it should be evaluated in terms of gestational age (GA), not birth weight (BW), because placental transfer of most nutrients from mother to fetus is dependent on GA, not BW. Judging from the limited data during the last 75 years, there was no significant correlation between GA and VE concentrations in circulation or in the red blood cells (RBCs), leukocytes, and buccal mucosal cells. In addition, the oxidizability of polyunsaturated fatty acids (PUFAs) in plasma or RBCs, as targets for protection by VE chain-breaking ability, was lower in preterm infants. However, because of the minimal information available about hepatic VE levels, which is considered a key determinant of whole body VE status, the decision on whether VE status of preterm infants is comparable with that of term infants should be postponed. Clinical trials of VE supplementation in preterm infants were repeatedly undertaken to investigate whether VE reduces severity or inhibits development of several diseases specific to preterm infants, namely retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), and germinal matrix hemorrhage - intraventricular hemorrhage (GMH-IVH). Most of these trials resulted in a misfire, with a few exceptions for IVH prevention. However, almost all these studies were performed from 1980s to early 1990s, in the pre-surfactant era, and the study populations were composed of mid-preterm infants with GAs of approximately 30 weeks (wks). There is considerable difference in ‘preterm infants’ between the pre- and post-surfactant eras; modern neonatal medicine mainly treats preterm infants of 28 wks GA or less. Therefore, these results are difficult to apply in modern neonatal care. Before considering new trials of VE supplementation, we should fully understand modern neonatal medicine, especially the recent method of oxygen supplementation. Additionally, a deeper understanding of recent progress in pathophysiology and therapies for possible target diseases is necessary to decide whether VE administration is still worth re-challenging in modern neonatal intensive care units (NICUs). In this review, we present recent concepts and therapeutic trends in ROP, BPD, and GMH-IVH for those unfamiliar with neonatal medicine. Numerous studies have reported the possible involvement of reactive oxygen species (ROS)-induced damage in relation to supplemental oxygen use, inflammation, and immature antioxidant defense in the development of both BPD and ROP. Various antioxidants effectively prevented the exacerbation of BPD and ROP in animal models. In the future, VE should be re-attempted as a complementary factor in combination with various therapies for BPD, ROP, and GMH-IVH. Because VE is a natural and safe supplement, we are certain that it will attract attention again in preterm medicine.
Article
Full-text available
Modern medicine has been waging a war on cancer for nearly a century with no tangible end in sight. Cancer treatments have significantly progressed, but the need to increase specificity and decrease systemic toxicities remains. Early diagnosis holds a key to improving prognostic outlook and patient quality of life, and diagnostic tools are on the cusp of a technological revolution. Nanotechnology has steadily expanded into the reaches of cancer chemotherapy, radiotherapy, diagnostics, and imaging, demonstrating the capacity to augment each and advance patient care. Nanomaterials provide an abundance of versatility, functionality, and applications to engineer specifically targeted cancer medicine, accurate early-detection devices, robust imaging modalities, and enhanced radiotherapy adjuvants. This review provides insights into the current clinical and pre-clinical nanotechnological applications for cancer drug therapy, diagnostics, imaging, and radiation therapy.
Excipients serve as vehicles, preservatives, solubilizers, and colorants for drugs, food, and cosmetics. They are considered to be inert at biological targets; however, several reports suggest that some could interact with human targets and cause unwanted effects. We investigated 40 commonly used drug excipients for cellular stress in the AsedaSciences® SYSTEMETRIC® Cell Health Screen, which was developed to estimate toxicity risk of small molecular entities (SMEs). The screen uses supervised machine learning (ML) to classify test compound cell stress phenotypes relative to a training set of on-market and withdrawn drugs. While 80% (n = 32) of the excipients did not show elevated risk in a broad, but pharmacologically relevant, concentration range (5 nM to 100 μM), we identified 20% (n = 8) with elevated risk. This group included two mercury containing preservatives, propyl gallate, methylene blue, benzethonium chloride, and cetylpyridinium chloride, all known for previously reported safety issues. All compounds were tested in parallel in an in vitro assay panel regularly used to investigate off-target effects of drug candidates. Target engagement in this assay panel confirmed risk-indicative biological activity for the same excipients, except propyl gallate, which may have a separate, interesting mechanism. We conclude that the SYSTEMETRIC Cell Health Screen, in conjunction with in vitro pharmacological profiling, can provide a fast and cost effective methodology for first line testing of SMEs, including excipients, to avoid cellular damage, particularly in the GI, where they are represented in high concentrations.
Article
Microemulsion systems have garnered tremendous interest in the pharmaceutical sector for a variety of drug delivery applications. Non‐ionic surfactants are often the preferred surfactant class given their uncharged nature, enhanced oral safety profile, and generally regarded as safe status as compared to other surfactant classes (Myers, Surfactant science and technology, 2005, p. 29), (Malmsten, Handbook of microemulsion science and technology, 1999, p. 755), (Grove & Mullertz, Chapter 5‐liquid self‐microemulsifying drug delivery systems, 2007), (Liu et al., Water‐insoluble drug formulation, 2008), (Hauss, Advanced Drug Delivery Reviews, 2007, 59, pp. 667–676), (Balazs, Solubility, delivery and ADME problems of drugs and drug‐candidates, 2011, p. 68). In this work, the phase behavior and microemulsion formation potential of four commonly used non‐ionic surfactants, PEG‐40 hydrogenated castor oil, Poloxamer 188, Polysorbate 80, and d‐α‐tocopherol polyethylene glycol succinate were studied via ternary phase diagram (TPD) mapping using a medium chain triglyceride, Miglyol 812. Results indicated notable differences in phase behavior despite similarities in hydrophilic–lipophilic balance value (13–15). All surfactants produced Winsor Type I, oil‐in‐water microemulsions at water concentrations above 40% wt/wt. Winsor Type II water‐in‐oil microemulsions were difficult to obtain even at high oil concentrations of ≥70% wt/wt. Winsor III microemulsions, though rare, were generally obtained in the middle regions of the TPD between 10% and 30% wt/wt water while Winsor IV microemulsions dominated at high surfactant concentrations of ≥45% wt/wt. Opaque emulsion areas were particularly notable in wax state surfactants. Polysorbate 80 and PEG‐40 hydrogenated castor oil demonstrated a high degree of synergism as well as the largest oil‐in‐water (o/w) and water‐in‐oil (w/o) microemulsion formation potential rendering them suitable for a number of enteral and parenteral applications.
Article
As the most popular microcontroller, Arduino is gaining interest for prototyping and optimizing instruments. Here, we report for the first time the use of Arduino as a standalone instrument to measure cell barrier integrity. Specifically, a transendothelial/epithelial resistance (TEER) meter was fabricated with an Arduino unit. TEER is an essential biophysical measurement to evaluate the integrity of biological barriers. Characterization of the device showed that the meter could accurately measure TEERs between 132 and 82,500 Ω·cm² with <3% errors, which covers the typical TEER ranges. The temporal resolution, measurement duration, and electrode configurations can be customized to meet a broad range of experimental designs. We have successfully applied the meter to measure the TEERs of endothelial cell monolayers, finding that cells treated with histamine had lower TEER values compared to untreated cells (793.4 ± 190.5 Ω·cm² vs. 3027.5 ± 664.4 Ω∙cm²; p < 0.001), which were validated and consistent with literature standards. In conclusion, we invented a low-cost Arduino-based TEER sensor capable of accurately measuring TEERs in relevant biological ranges. We also included detailed tutorials in the supplementary information to promote translation of the technology.
Article
The lipophilic character of peptides can be tremendously improved by hydrophobic ion pairing (HIP) with counterions to be efficiently incorporated into lipid-based nanocarriers (NCs). Herein, HIPs of exenatide with the cationic surfactant tetraheptylammonium bromide (THA) and the anionic surfactant sodium docusate (DOC) were formed to increase its lipophilicity. These HIPs were incorporated into lipid based NCs comprising 41% Capmul MCM, 15% Captex 355, 40% Cremophor RH and 4% propylene glycol. Exenatide-THA NCs showed a log D lipophilic phase (LPh)/release medium (RM) of 2.29 and 1.92, whereas the log D LPh/RM of exenatide-DOC was 1.2 and -0.9 in simulated intestinal fluid and Hanks’ balanced salts buffer (HBSS), respectively. No significant hemolytic activity was induced at a concentration of 0.25% (m/v) of both blank and loaded NCs. Exenatide-THA NCs and exenatide-DOC NCs showed a 10-fold and 3-fold enhancement in intestinal apparent membrane permeability compared to free exenatide, respectively. Furthermore, orally administered exenatide-THA and exenatide-DOC NCs in healthy rats resulted in a relative bioavailability of 27.96 ± 5.24% and 16.29 ± 6.63%, respectively, confirming the comparatively higher potential of the cationic surfactant over the anionic surfactant. Findings of this work highlight the potential of the type of counterion used for HIP as key to successful design of lipid-based NCs for oral exenatide delivery.
Article
The application of hot melt coating (HMC) as an economic and solvent-free technology is restricted in pharmaceutical development, due to the instable solid-state of HMC excipients resulting in drug release instability. We have previously introduced polyglycerol esters of fatty acids (PGFAs) with stable solid-state (Part 1). In this work we showed a novel application of PGFAs as HMC excipients with stable performance. Three PGFA compounds with a HLB range of 5.1 to 6.2 were selected for developing immediate-release formulations. The HMC properties were investigated. The viscosity of molten lipids at 100°C was suitable for atomizing. The DSC data showed the absence of low solidification fractions, thus reduced risk of agglomeration during the coating process. The driving force for crystallization of selected compounds was lower and the heat flow exotherms were broader compared to conventional HMC formulations, indicating a lower energy barrier for nucleation and lower crystallization rate. Lower spray rates and a process temperature close to solidification temperature were desired to provide homogeneous coating. DSC and X-ray diffraction data revealed stable solid state during 6 months storage at 40°C. API release was directly proportional to HLB and indirectly proportional to crystalline network density and was stable during investigated 3 months. Cytotoxicity was assessed by dehydrogenase activity and no in vitro cytotoxic effect was observed.
Article
A promising strategy for treatment of EGFR-dependent tumours is EGFR signal transduction suppression via inhibition of HMG-CoA reductase using high doses of statins, popular cholesterol-lowering drugs. The main purpose of this study was to obtain targeted long circulating immunoliposomes containing simvastatin (tLCLS) with anti-EGFR antibody attached to their surface and to test whether they can be effective in treatment of TNBC. The designed tLCLS were characterized in terms of physicochemical properties and long-term stability. In vitro experiments conducted on MDA-MB-231 cells demonstrated that tLCLS induced apoptosis and are characterized by IC50 of 7.5 µM. Treatment of studied cells with tLCLS led to a decrease in membrane order and inhibited PI3K/Akt signalling. Analyses of efficacy of the tLCLS in in vivo experiments in model animals indicate that immunoliposomes were effectively delivered to tumours. Our results showed that regardless of whether tLCLS were administered before or after tumour formation, at the tested dose they inhibited tumour growth by an average of 25% in comparison to the control. However, the results were not statistically significant. The experiments described above allowed us to test the possibility of using immunoliposomes as simvastatin carriers delivering increased amounts of the drug to tumour cells.
Article
The first fully developed herbal medicine in Brazil, Acheflan®, contains the essential oil of Cordia verbenacea DC that is present at 0.5% in both a cream and aerosol form. Its topical use is indicated for the treatment of muscle aches and tendinitis to alleviate discomfort related to associated inflammatory processes. The two principal components of the essential oil involved in the anti-inflammatory activity are α-humulene and (-)-trans-caryophyllene, which need to penetrate the skin to exert their effects. The objective of this work was to evaluate, for the first time, the in vitro release profiles and the permeation of these commercial phytotherapeutics using Franz diffusion cells. The results indicate that the commercial products have a low release profile that appears to restrict permeation of the oil components. This was in contrast to the pure essential oil, which presented a good capacity to permeate through skin. To improve release, new nanoemulsion formulations were developed and evaluated for their release profiles, permeation and kinetic parameters. The transition to a nanoemulsion system was shown to improve the permeation profiles of the essential oil as these formulations had a high in vitro permeation and release flux rate. In addition, the final formulation presented an adequate droplet distribution size, polydispersity index and pH. Overall, we observed that the composition of formulations that contain Cordia verbenacea DC can directly influence the diffusion of the oil components through dermal barriers, which can potentiate their intended anti-inflammatory effects.
Article
Amphipathic, non-ionic, surfactants are widely used in pharmaceutical, food, and agricultural industry to enhance product features; as pharmaceutical excipients they are also aimed at increasing cell membrane permeability and consequently improving e.g. oral drugs absorption. Here we report on the concentration- and time-dependent succession of events occurring throughout, and subsequent exposure of Caco-2 epithelium to a ‘typical’ non-ionic surfactant (Kolliphor® HS15) to provide a molecular explanation for non-ionic surfactant cytotoxicity. The study shows that the conditions of surfactant exposure, which increase cell membrane fluidity and permeability, produced rapid (within 5 minutes) redox and mitochondrial effects. Apoptosis was triggered early during exposure (within 10 minutes), and relied upon an initial mitochondrial membrane hyperpolarization (5-10 minutes) as a crucial step, leading to its subsequent depolarization and caspase-3/7 activation (60 minutes). The apoptotic pathway appears to be triggered prior to substantial surfactant-induced membrane damage (observed ≥60 minutes). We hence propose that the cellular response to the model non-ionic surfactant is triggered via surfactant-induced increase in plasma membrane fluidity - a phenomenon akin to the stress response to membrane fluidization induced by heat shock - and consequent apoptosis. Therefore, the fluidization effect that confers surfactants the ability to enhance drug permeability may also be intrinsically linked to the propagation of their cytotoxicity. The reported observations have important implications for the safety of a multitude of non-ionic surfactants used in drug delivery formulations and, also to other permeability enhancing compounds with similar plasma membrane fluidizing mechanisms.
Article
Full-text available
The aim of this work was to prepare and optimize a solid self-nanoemulsifying drug delivery system pre-concentrate (SSP) containing water-insoluble flurbiprofen (FL) using a novel pseudo-ternary phase diagram. The pseudo-ternary phase diagram, composed of FL as the drug and dispersion core, Kollisolv MCT 70 as the oil phase, and TPGS (tocopherol polyethylene glycol 1000 succinate) as the surfactant, was constructed for the determination of the SSP region. SSP was investigated in terms of particle size, physical state by differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD), in vitro dissolution and oral pharmacokinetics in rats. The determined SSP (FL/Kollisolv MCT 70/TPGS = 10/10/80, weight %) in the pseudo-ternary phase diagram had the melting point of 32.37 °C and uniform mean particle size of below 30 nm without any precipitation of FL in the dispersion. In the dissolution test, the SSP exhibited 95.70 ± 3.40% of release at 15 min, whereas the raw FL showed poor dissolution (i.e., 6.75 ± 1.30%) at that time point. In addition, the SSP showed the enhanced oral absorption (i.e., 1.93-fold increase in AUCinfinite) as compared to the suspension group of raw FL. Therefore, the developed SSP would be a promising drug delivery system with excellent solubilization, dissolution, and bioavailability for FL.
Article
Vitamin E is obtained only through the diet and has a number of important biological activities, including functioning as an antioxidant. Evidence that free radicals may contribute to pathological processes such as bronchopulmonary dysplasia (BPD), a disease of prematurity associated with increased lung injury, inflammation and oxidative stress, led to trials of the antioxidant vitamin E (α-tocopherol) to prevent BPD with variable results. These trials were all conducted at supraphysiologic doses and 2 of these trials utilized a formulation containing a potentially harmful excipient. Since 1991, when the last of these trials was conducted, both neonatal management strategies for minimizing oxygen and ventilator-related lung injury and our understanding of vitamin E isoforms in respiratory health have advanced substantially. It is now known that there are differences between the effects of vitamin E isoforms α-tocopherol and γ-tocopherol on the development of respiratory morbidity and inflammation. What is not known is whether improvements in physiologic concentrations of individual or combinations of vitamin E isoforms during pregnancy or following preterm birth might prevent or reduce BPD development. The answers to these questions require adequately powered studies targeting pregnant women at risk of preterm birth or their premature infants immediately following birth, especially in certain subgroups that are at increased risk of vitamin E deficiency (e.g., smokers). The objective of this review is to compile, update, and interpret what is known about vitamin E isoforms and BPD since these first studies were conducted, and suggest future research directions.
Article
Full-text available
Cremophor EL (CrEL) is a formulation vehicle used for various poorly-water soluble drugs, including the anticancer agent paclitaxel (Taxol). In contrast to earlier reports, CrEL is not an inert vehicle, but exerts a range of biological effects, some of which have important clinical implications. Its use has been associated with severe anaphylactoid hypersensitivity reactions, hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral neuropathy. The pharmacokinetic behaviour of CrEL is dose-independent, although its clearance is highly influenced by duration of the infusion. This is particularly important since CrEL can affect the disposition of various drugs by changing the unbound drug concentration through micellar encapsulation. In addition, it has been shown that CrEL, as an integral component of paclitaxel chemotherapy, modifies the toxicity profile of certain anticancer agents given concomitantly, by mechanisms other than kinetic interference. A clear understanding of the biological and pharmacological role of CrEL is essential to help oncologists avoid side-effects associated with the use of paclitaxel or other agents using this vehicle. With the present development of various new anticancer agents, it is recommended that alternative formulation approaches should be pursued to allow a better control of the toxicity of the treatment and the pharmacological interactions related to the use of CrEL.
Article
Full-text available
Immunosafety analysis of pharmaceutical surfactants is an important step in understanding the complex mechanisms by which they induce side effects in susceptible patients. This paper provides experimental evidences that polyethoxylated surfactants, Cremophor-EL and Tween-80, also known as Polysorbate-80, activate the complement system in vitro, in normal human serum and plasma. They appeared to be more efficient reactogens than their structural homolog, Tween-20. Cremophor-EL and Tween-80 promoted the generation of biologically active complement products, C3a, C5a and C5b-9. Consistently, Paclitaxel and Taxotere (Docetaxel), pharmaceuticals formulated in Cremophor-EL and Tween-80, activated the complement system in similar extent. Moreover, comparison of serum reactivity against the drug-loaded and drug-free formulations exhibited a significant linear correlation. Taken together, these results are consistent with the hypothesis that therapeutic side effects, such as acute hypersensitivity and systemic immunostimulation, caused by intravenous nanomedicines containing polyethoxylated detergents such as Cremophor-EL and Tween-80, can be attributed to complement activation-derived inflammatory mediators.
Article
Full-text available
Nystatin is commonly employed to treat fungal infections in the mouth. It is not absorbed via the stomach and it will therefore not treat fungal infections in any part of the body other than the mouth. Nystatin buccoadhesive tablets release the drug very slowly due to the poor solubility of nystatin in water and also the presence of polymers with mucoadhesive properties. Therefore, the aim of the present study was to improve drug release from buccoadhesive tablets, while retaining adequate mucoadhesive properties. To this end, a solid dispersion of nystatin: lactose (1:3) was prepared and mixed with xanthan. The effects of hydrophilic surfactants such as cremophor RH40 and Tween 80 on drug release and mucoadhesive properties of nystatin tablets were also investigated as were swelling and erosion indices and strength of bioadhesion in vitro to a biological membrane. The interaction between nystatin and lactose in solid dispersion formulation was investigated by XRPD, FT-IR and DSC. The results showed that a solid dispersion formulation and mucoadhesive tablets containing surfactants led to faster drug release than their simple physical mixtures. Drug release was also faster from a solid dispersion compared to tablets containing surfactants. Swelling and erosion results showed that tablets made of a solid dispersion swelled and eroded faster than a physical mixture formulation. The presence of surfactant slightly increased the degree of swelling and erosion of buccoadhesive tablets.
Article
Full-text available
In recent years, a new cell-based high throughput paradigm has emerged, which seeks to identify novel, pharmacologically active cytoprotective compounds. The essence of this approach is to create experimental models of cell injury relevant for a particular disease by establishing in vitro cell-based models, followed by high-throughput testing of compounds that affect the cellular response in a desired manner. Prior approaches typically used simple end-point analyses. To assess the cytoprotective effects of novel drug candidates in real-time, we have applied a cell-microelectronic sensing technique (RT-CES), which measures changes in the impedance of individual microelectronic wells that correlates linearly with cell index (reflecting cell number, adherence and cell growth), thereby allowing the continuous determination of cell viability during oxidative stress. In vitro cytotoxicity was elicited by hydrogen peroxide in myocytes (H9c2) and hepatocytes (Hep3B). Cells were post-treated at 30 min with various reference molecules and novel cytoprotective compounds. Cytoprotection detected in the RT-CES system correlated well with the results of two classical end-point-based methods (improvement in MTT and reduction of LDH release). The RT-CES method, when used as described in the current report, is suitable for the screening of molecular libraries to identify molecules or molecule combinations that attenuate oxidative stress-induced cell damage.
Article
Full-text available
The ability of a human colonocyte epithelial cell line (CaCo-2) to synthesise leukotriene B4 was examined. In addition, the effects of stimulation with calcium ionophore, inhibition by a drug which specifically prevents activation of 5-lipoxygenase, and modification of the fatty acid composition of membrane phospholipids on leukotriene B4 synthesis were assessed. Incubation with calcium ionophore (A23187) resulted in a dose and time dependent increase in leukotriene B4 synthesis. After cell phospholipids had been enriched with oleic acid, linoleic acid, and arachidonic acid, leukotriene B4 synthesis was found to be increased 3.2-fold, 5.5-fold, and 6.1-fold above control. Treatment with MK-886 inhibited leukotriene B4 synthesis by 79% to 94% in the various groups. Variations in the polyunsaturated fatty acid content of intestinal epithelial cells could be important in the modulation of cellular responses. We have shown for the first time in this human intestinal epithelial cell its ability to synthesise leukotriene B4. In addition, leukotriene B4 synthesis can be modulated by the fatty acid composition of membrane phospholipids, which can be altered by dietary fatty acids. The synthesis of chemotatic factors, such as leukotriene B4, by the mucosal epithelium may contribute to the recruitment of granulocytes into the colonic mucosa and across the epithelium, giving rise to the crypt abscesses which characterise ulcerative colitis.
Article
Full-text available
Cremophor EL (CrEL) is a formulation vehicle used for various poorly-water soluble drugs, including the anticancer agent paclitaxel (Taxol). In contrast to earlier reports, CrEL is not an inert vehicle, but exerts a range of biological effects, some of which have important clinical implications. Its use has been associated with severe anaphylactoid hypersensitivity reactions, hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral neuropathy. The pharmacokinetic behaviour of CrEL is dose-independent, although its clearance is highly influenced by duration of the infusion. This is particularly important since CrEL can affect the disposition of various drugs by changing the unbound drug concentration through micellar encapsulation. In addition, it has been shown that CrEL, as an integral component of paclitaxel chemotherapy, modifies the toxicity profile of certain anticancer agents given concomitantly, by mechanisms other than kinetic interference. A clear understanding of the biological and pharmacological role of CrEL is essential to help oncologists avoid side-effects associated with the use of paclitaxel or other agents using this vehicle. With the present development of various new anticancer agents, it is recommended that alternative formulation approaches should be pursued to allow a better control of the toxicity of the treatment and the pharmacological interactions related to the use of CrEL.
Article
Full-text available
A review of commercially available oral and injectable solution formulations reveals that the solubilizing excipients include water-soluble organic solvents (polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide), non-ionic surfactants (Cremophor EL, Cremophor RH 40, Cremophor RH 60, d-alpha-tocopherol polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, Solutol HS 15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire 44/14, Softigen 767, and mono- and di-fatty acid esters of PEG 300, 400, or 1750), water-insoluble lipids (castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil and palm seed oil), organic liquids/semi-solids (beeswax, d-alpha-tocopherol, oleic acid, medium-chain mono- and diglycerides), various cyclodextrins (alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and sulfobutylether-beta-cyclodextrin), and phospholipids (hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, L-alpha-dimyristoylphosphatidylcholine, L-alpha-dimyristoylphosphatidylglycerol). The chemical techniques to solubilize water-insoluble drugs for oral and injection administration include pH adjustment, cosolvents, complexation, microemulsions, self-emulsifying drug delivery systems, micelles, liposomes, and emulsions.
Article
Full-text available
Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.
Article
BACKGROUND Previous studies indicate that Cremophor EL (CEL), the excipient for Taxol, a clinical preparation of paclitaxel, has biologic properties per se.METHODS The cytotoxic activity of Taxol and its solvents CEL/ethanol, paclitaxel in ethanol, and 14 other cytotoxic drugs was investigated in vitro in 10 human carcinoma cell lines and 183 tumor samples from patients with tumors of various types. Cytotoxicity was determined by the fluorometric microculture cytotoxicity assay.RESULTSIn the cell lines, Taxol was generally more active than paclitaxel; this may have been due to an additive effect of the diluent. This activity was pronounced in sublines expressing tubulin-associated and P-glycoprotein-mediated drug resistance, indicating involvement of these mechanisms in paclitaxel resistance and their modulation by CEL. Taxol and paclitaxel were highly cross-resistant to other tubulin-active agents, whereas the low cytotoxic effect of CEL seemed unrelated to other drugs. In the samples from patients, Taxol was less active than in the cell lines but showed a differential activity that corresponded reasonably well with that in the clinic. CEL and Taxol were similarly active, indicating that paclitaxel did not add substantially to the activity of Taxol.CONCLUSIONS Whereas the cell line data clearly confirmed the well-known properties of paclitaxel, a more valid model using tumor cells from patients demonstrated that CEL significantly contributes to the efficacy of Taxol in vitro. The clinical relevance of this finding remains to be elucidated. Cancer 1997; 79:1225-33. © 1997 American Cancer Society.
Article
The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood–brain barrier.
Article
Sucrose esters are effective solubilizers and there is an interest to use them as pharmaceutical excipients for nasal drug delivery. We have determined for the first time the non-toxic doses of laurate and myristate sucrose esters by four independent methods, and their effects on epithelial permeability using RPMI 2650 human nasal epithelial cell line. Based on real-time cell electronic sensing, MTT dye conversion and lactate dehydrogenase release methods reference surfactant Cremophor RH40 proved to be the least toxic excipient, and could be used at 5mg/mL concentration for 1h in epithelial cells without cellular damage. The non-toxic dose of Tween 80 was 1 mg/mL, while the dose of laurate and myristate sucrose esters that could be safely used on cells for 1 h was 0.1 mg/mL. Both the reference surfactants and the sucrose esters significantly enhanced the permeability of epithelial cell layers for the paracellular marker FITC-labelled 4.4 kDa dextran at 0.1 mg/mL concentration. The effects of sucrose esters on epithelial permeability were dose-dependent. These data indicate that laurate and myristate sucrose esters can be potentially used as permeability enhancers in nasal formulations to augment drug delivery to the systemic circulation.
Article
Oxymatrine (OMT), a water-soluble drug, has a very low oral bioavailability because of its low membrane permeability and its biotransformation in the gastrointestinal tract. Formulated as an oxymatrine-phospholipid complex (OMT-PLC) can improve the lipid solubility and effectiveness of OMT. The purpose of this study was to explore the utility of the combination of a microemulsion and an OMT-PLC as a topical delivery vehicle for enhancing the absorption and efficacy of OMT. The solubility of OMT-PLC was determined and phase diagrams of microemulsions were constructed. Various microemulsion formulations were developed and characterized by their physicochemical properties, and their in vitro and in vivo permeability through skin. An optimal microemulsion (ME4), which presented as spherical droplets and consisted of 10.0% OMT-PLC, 8.0% isopropyl myristate, 30.0% Cremophor RH40/polyethylene glycol 400 (1:1) and 52.0% water, was selected. It possessed an average droplet size of 32.4 nm, a low viscosity of 113.7 mPa · s, and a high cloud point of 88°C. Compared to the control solution, ME4 provided better skin permeability in vitro and a higher retention ratio of OMT in skin in vivo. Moreover, ME4 significantly enhanced the antiproliferative activity of OMT on scar fibroblasts. These results indicate that the combination of a microemulsion and a phospholipid complex represents an effective vehicle for topical delivery of OMT.
Article
The formation of lipid digestion products is a relevant parameter for effective in vivo performance of self-emulsifying drug delivery systems (SEDDS). These lipid-based formulations contain non-ionic surfactants, therefore the effects of polysorbate 80 (PS 80), D-alpha-tocopheryl polyethylene glycol (1000) succinate (TPGS), Surfhope((R)) sugar ester D-1216 (sucrose laurate), Cremophor EL (Cr EL) and Cremophor RH 40 (Cr RH 40) on triglyceride digestion by pancreatic lipases were tested in vitro using olive oil as model substrate. The IC(50) determined are 0.13mM (PS 80), 0.08mM (TPGS), 0.46mM (sucrose laurate), 0.19mM (Cr EL) and 0.04mM (Cr RH 40). The extent of inhibitory action is ranked in downward order: Cremophor RH 40>TPGS>polysorbate 80>Cremophor EL>sucrose laurate. The effects already occur below the critical micelle concentrations (CMC) of the detergents. At low concentrations of polysorbate 80 the inhibition shows a competitive mechanism. Furthermore, degradation by pancreatic enzymes of the surfactants themselves had been tested which also can affect the solubilization capacity of the intestinal fluid after administration of a SEDDS. Polysorbate 80, Cremophor EL and Cremophor RH 40 were shown to be susceptible to digestion by pancreatic enzymes. Fatty acid esters of polysorbate 80 were hydrolyzed with an extent of 14% (+/-1.0%). 14.4% (+/-3.3%) in case of Cremophor EL had been hydrolyzed, whereas Cremophor RH 40 had been degraded to a lower extend of 6.1% (+/-2.8%). TPGS and sucrose laurate appeared to be stable in present of pancreatic enzymes.
Article
A novel non-ionic surfactant, peptoad G, is evaluated for its solubilizing capacity and cytotoxicity in order to explore its possible use in aqueous formulation of hydrophobic drugs. Solubility studies were carried out using ten model hydrophobic drugs, and cytotoxicity of the surfactant was evaluated in three different cell lines using the MTT assay. It was shown that peptoad G enhances the solubility of the ten model drugs to different extents, ranging from 20- to 1100-fold, which correlated with the number of hydrogen-bonding sites on the drug molecules. The in vitro cytotoxicity studies revealed comparable cytotoxicity of peptoad G to that of cremophor EL. The results suggest peptoad G possesses potential as an alternative to conventional solubilizers in hydrophobic drug formulations.
Article
Intranasal administration of molecules has been investigated as a non-invasive way for delivery of drugs to the brain in the last decade. Circumvention of both the blood-brain barrier and the first-pass elimination by the liver and gastrointestinal tract is considered as the main advantages of this method. Because of the rapid mucociliary clearance in the nasal cavity, bioadhesive formulations are needed for effective targeting. Our goal was to develop a formulation containing sodium hyaluronate, a well-known mucoadhesive molecule, in combination with a non-ionic surfactant to enhance the delivery of hydrophilic compounds to the brain via the olfactory route. Fluorescein isothiocyanate-labeled 4 kDa dextran (FD-4), used as a test molecule, was administered nasally in different formulations to Wistar rats, and detected in brain areas by fluorescent spectrophotometry. Hyaluronan increased the viscosity of the vehicles and slowed down the in vitro release of FD-4. Significantly higher FD-4 transport could be measured in the majority of brain areas examined, including olfactory bulb, frontal and parietal cortex, hippocampus, cerebellum, midbrain and pons, when the vehicle contained hyaluronan in combination with absorption enhancer. The highest concentrations of FD-4 could be detected in the olfactory bulbs, frontal and parietal cortex 4h after nasal administration in the mucoadhesive formulation. Intravenous administration of a hundred times higher dose of FD-4 resulted in a lower brain penetration as compared to nasal formulations. Morphological examination of the olfactory system revealed no toxicity of the vehicles. Hyaluronan, a non-toxic biomolecule used as a mucoadhesive in a nasal formulation, increased the brain penetration of a hydrophilic compound, the size of a peptide, via the nasal route.
Article
Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.
Article
This investigation was undertaken to develop cytotoxicity assay systems using primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate oculotoxic agents and the ability of these in vitro assay systems to predict irritancy potential and delayed toxicity. We have characterized the epithelial nature of the cultures by identifying keratins with antikeratin antibodies (AE1/AE3) and by demonstrating metabolic enzymes important to the integrity of the cells: lactate dehydrogenase, glucose 6-phosphate dehydrogenase and aldolase. Eight surfactants were compared and ranked according to their cytotoxic potential. We evaluated cytotoxicity by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium, by making morphological observations and by assessing lysosomal neutral red uptake and mitochondrial 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. The cells were treated for 1 h with the surfactants and the possibility of delayed toxicity was evaluated 24 h after removal of the surfactant. The cytotoxicity of the different types of surfactants as shown by all the tests was cationic > anionic = amphoteric > non-ionic. Triton X-100, a non-ionic surfactant but a severe irritant, had a ranking similar to anionic surfactants. The in vitro rankings corresponded well to reported in vivo Draize rabbit eye test data. The 24-h test for lactate dehydrogenase leakage showed that mild and non-irritating surfactants did not demonstrate any subsequent damage after a 1-h exposure, but the extreme and severe surfactants continued to show further damage after the 1-h exposure. These in vitro findings were similar to reported in vivo results. The neutral red and MTT tests did not adequately predict the prolonged toxicity of the more irritating surfactants, as was demonstrated by the lactate dehydrogenase leakage test. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to rank the cytotoxic potential of surfactants, but only the lactate dehydrogenase leakage test was able to assess prolonged cell injury.
Article
Nervous system complications resulting from i.v. administration of cyclosporine (CS) are especially frequent in liver transplant recipients. Because CS is insoluble in water, the i.v. preparation is formulated in a polyoxyethylated castor oil and ethyl alcohol. Rat dorsal root ganglion neurons exposed in vitro to the i.v. preparation exhibited axonal swelling and degeneration. No effect of CS (dissolved directly in serum) was seen on testing individual components of the i.v. solution. However, 0.1% polyoxyethylated castor oil (volume of solute/volume of solvent) produced axonal swelling and degeneration and 0.001% polyoxyethylated castor oil produced demyelination in vitro. Polyoxyethylated castor oil is manufactured by reacting castor oil with ethylene oxide, and we speculate that residual ethylene oxide or a polymerization product may be responsible for the in vitro neurotoxicity. Although little is known about the pharmacokinetics of polyoxyethylated castor oil, plasma levels of 0.001 to 0.01% polyoxyethylated castor oil (volume of solute/volume of solvent) are probably achieved with therapeutic doses of the i.v. CS preparation.
Article
Cyclosporin A (CsA) is widely used as the immunosuppressant of choice for preventing graft rejection. However, its clinical use is hampered by certain side effects, especially its nephrotoxicity and other cardiovascular side effects. CsA for intravenous infusion contains cremophor (Cre) and this vehicle has significant adverse effects on endothelial function and vascular muscle. The present study was aimed at investigating the direct effects of CsA and Cre on isolated and perfused rat hearts in the dosage that closely approximates the peak level achieved for the prevention of graft rejection in the rat. Transplantation is a clinical setting in which the myocardium may be exposed to transient ischemia. In this study, we have shown that the vehicle of CsA, namely Cre, has significant adverse effects on cardiac function. We observed a reduction in coronary flow and aortic output. Addition of CsA appeared to induce a further reduction of aortic flow. We have also shown that a significant increase of thiobarbituric acid reactive substances, considered as an index of lipid peroxidation, occurred in the reperfused heart in the presence of Cre+CsA. Our experimental study shows that Cre turned out to be toxic to myocardium by itself. In the heart, potential Cre-CsA interactions possibly potentiating CsA toxicity could not be excluded. The increase of lipid peroxidation in the heart perfused with CsA suggests that reactive oxygen species may be involved in the detrimental effects of this substance on the heart.
Article
Previous studies indicate that Cremophor EL (CEL), the excipient for Taxol, a clinical preparation of paclitaxel, has biologic properties per se. The cytotoxic activity of Taxol and its solvents CEL/ethanol, paclitaxel in ethanol, and 14 other cytotoxic drugs was investigated in vitro in 10 human carcinoma cell lines and 183 tumor samples from patients with tumors of various types. Cytotoxicity was determined by the fluorometric microculture cytotoxicity assay. In the cell lines, Taxol was generally more active than paclitaxel; this may have been due to an additive effect of the diluent. This activity was pronounced in sublines expressing tubulin-associated and P-glycoprotein-mediated drug resistance, indicating involvement of these mechanisms in paclitaxel resistance and their modulation by CEL. Taxol and paclitaxel were highly cross-resistant to other tubulin-active agents, whereas the low cytotoxic effect of CEL seemed unrelated to other drugs. In the samples from patients, Taxol was less active than in the cell lines but showed a differential activity that corresponded reasonably well with that in the clinic. CEL and Taxol were similarly active, indicating that paclitaxel did not add substantially to the activity of Taxol. Whereas the cell line data clearly confirmed the well-known properties of paclitaxel, a more valid model using tumor cells from patients demonstrated that CEL significantly contributes to the efficacy of Taxol in vitro. The clinical relevance of this finding remains to be elucidated.
Article
In order to examine whether immunosuppressive treatment could be responsible for the reduced exercise capacity of heart transplant recipients (HTR), we studied the effects of long-term immunosuppressive treatment with cyclosporin A (CsA) and its vehicle (2/3 cremophor and 1/3 alcohol diluted in olive oil) on in situ mitochondrial respiration of different muscles. Rats were fed for 3 weeks with 10 or 25 mg/kg/day CsA in its vehicle (CsA10 and CsA25 groups), or vehicle or H(2)O. Oxygen consumption rate was measured in saponin skinned fibers without (V(0)) and with ADP until maximal respiration (V(max)) was reached and K(M)for ADP as well as V(max)were calculated using non-linear fit of the Michaelis-Menten equation. In the cardiac muscle of the CsA25 group, V(0)and V(max)were decreased by immunosuppressive treatment respectively from 6.33+/-0.51 to 3.18+/-0.3micromol O(2)/min/g dw (P<0.001) and from 29.0+/-1.5 to 18.1+/-1.6micromol O(2)/min/g dw (P<0.001), an effect which could be entirely attributed to the vehicle itself, with no difference between CsA10 and CsA25. Regulation of cardiac mitochondrial respiration by ADP was altered by vehicle with the K(M)for ADP decreasing from 371+/-37 to 180+/-21microm(P<0.001). A similar trend was observed in the diaphragm or soleus, although to a lesser extent. In contrast, V(0)and V(max)decreased in glycolytic gastrocnemius muscle respectively from 1.7+/-0.2 to 0.94+/-0.14 (P<0. 01) and from 6.8+/-0.3 to 5.1+/-0.4micromol O(2)/min/g dw (P<0.001) in the CsA25 group, but the main effects could be attributed to CsA itself. It was concluded that immunosuppressive treatment induces a deleterious effect on cardiac and skeletal muscle oxidative capacities, mainly due to cremophor, the main component of vehicle.
Article
Signal transduction is initiated by complex protein–protein interactions between ligands, receptors and kinases, to name only a few. It is now becoming clear that lipid micro-environments on the cell surface — known as lipid rafts — also take part in this process. Lipid rafts containing a given set of proteins can change their size and composition in response to intra- or extracellular stimuli. This favours specific protein–protein interactions, resulting in the activation of signalling cascades.
Article
A new simulation method, dissipative particle dynamics, is applied to model biological membranes. In this method, several atoms are united into a single simulation particle. The solubility and compressibility of the various liquid components are reproduced by the simulation model. When applied to a bilayer of phosphatidylethanolamine, the membrane structure obtained matches quantitatively with full atomistic simulations and with experiments reported in the literature. The method is applied to investigate the cause of cell death when bacteria are exposed to nonionic surfactants. Mixed bilayers of lipid and nonionic surfactant were studied, and the diffusion of water through the bilayer was monitored. Small transient holes are seen to appear at 40% mole-fraction C(9)E(8), which become permanent holes between 60 and 70% surfactant. When C(12)E(6) is applied, permanent holes only arise at 90% mole-fraction surfactant. Some simulations have been carried out to determine the rupture properties of mixed bilayers of phosphatidylethanolamine and C(12)E(6). These simulations indicate that the area of a pure lipid bilayer can be increased by a factor 2. The inclusion of surfactant considerably reduces both the extensibility and the maximum stress that the bilayer can withstand. This may explain why dividing cells are more at risk than static cells.
Article
P-glycoprotein (P-gp), a multidrug resistance (MDR) protein encoded by the MDR1 gene in humans, is responsible for the efflux of structurally diverse drugs. Previous studies in our laboratory have shown that excipients such as poly(ethylene)glycol (PEG)-300, Cremophor EL, and Tween 80 inhibit P-gp activity in Caco-2 cell monolayers. The objective of this study was to determine the effects of these excipients in an MDR1- transfected Madin Darby Canine Kidney (MDR1-MDCK) cell line and to compare the results with those obtained from Caco-2 cells. The results presented herein show that PEG-300 (20%, v/v) causes almost complete inhibition of P-gp activity in both Caco-2 and MDR1-MDCK cell monolayers, whereas Cremophor EL (0.1%, w/v) and Tween 80 (0.05%, w/v) only partially inhibit P-gp activity in Caco-2 cells. Cremophor EL (0.1%, w/v) and Tween 80 (0.05%, w/v) were inactive as P-gp inhibitors in MDR1-MDCK cell monolayers. This inability of Tween 80 and Cremphor EL to inhibit P-gp activity in MDR1-MDCK cells may be related to differences in the interactions of the surfactants with these different cell membranes. PEG-induced changes in P-gp activity are probably related to changes in the fluidity of the polar head group regions of cell membranes.
Article
The pharmacological testing of natural products can often be hampered by the poor solubility of such compounds in non-toxic solvents. There is thus a need for a suitable agent for solubilization of natural substances to allow testing on a variety of cell lines in-vitro. Such an agent should ideally have no direct effects on any of the commonly used cell lines from a variety of tissues and mammalian species to allow proper comparison. In this study, the lichen metabolite (+)-usnic acid, a dibenzofuran derivative, was used as a prototype for an insoluble natural product with the aim of finding a solvent that was both capable of solubilizing usnic acid and was free of direct activity against a test cell line. Solubilization was measured at different pH values in various concentrations of co-solvents (glycofurol 75, propylene glycol, polyethylene glycol 400), surfactants (polysorbate 20 and Cremophor RH40), and the complexing agent 2-hydroxypropyl-beta-cyclodextrin. The solubility achieved in a 20% aqueous solution was 0.11 mg mL(-1) for propylene glycol, 0.19 for PEG 400, 0.27 for glycofurol 75, 0.57 for Cremophor RH40, 0.68 for 2-hydroxypropyl-beta-cyclodextrin and 0.84 for polysorbate 20. The direct effects of the various solvent systems were tested on the human leukaemia cell line K-562 in a standard proliferation assay. Most of the solvents proved toxic with the exception of propylene glycol, PEG 400 and 2-hydroxypropyl-beta-cyclodextrin. Anti-proliferative activity of usnic acid could be demonstrated with an ED50 (amount of substance required to reduce thymidine uptake to 50% of uptake by untreated control culture) of 4.7 microg mL(-1) using PEG 400 and 2-hydroxypropyl-beta-cyclodextrin but only the latter gave satisfactory solubility. 2-Hydroxypropyl-beta-cyclodextrin was thus identified as a solubilizing agent that fulfilled both set criteria of solubility and lack of toxicity against the test cells.
Article
The oral delivery of hydrophobic drugs presents a major challenge because of the low aqueous solubility of such compounds. Self-emulsifying drug delivery systems (SEDDS), which are isotropic mixtures of oils, surfactants, solvents and co-solvents/surfactants, can be used for the design of formulations in order to improve the oral absorption of highly lipophilic drug compounds. SEDDS can be orally administered in soft or hard gelatin capsules and form fine relatively stable oil-in-water (o/w) emulsions upon aqueous dilution owing to the gentle agitation of the gastrointestinal fluids. The efficiency of oral absorption of the drug compound from the SEDDS depends on many formulation-related parameters, such as surfactant concentration, oil/surfactant ratio, polarity of the emulsion, droplet size and charge, all of which in essence determine the self-emulsification ability. Thus, only very specific pharmaceutical excipient combinations will lead to efficient self-emulsifying systems. Although many studies have been carried out, there are few drug products on the pharmaceutical market formulated as SEDDS confirming the difficulty of formulating hydrophobic drug compounds into such formulations. At present, there are four drug products, Sandimmune and Sandimmun Neoral (cyclosporin A), Norvir (ritonavir), and Fortovase (saquinavir) on the pharmaceutical market, the active compounds of which have been formulated into specific SEDDS. Significant improvement in the oral bioavailability of these drug compounds has been demonstrated for each case. The fact that almost 40% of the new drug compounds are hydrophobic in nature implies that studies with SEDDS will continue, and more drug compounds formulated as SEDDS will reach the pharmaceutical market in the future.
Article
Cremophor EL, a surfactant for pharmaceutical products, augments the cytotoxicity of hydrogen peroxide in rat thymocytes [Iwase, K., Oyama, Y., Tatsuishi, T., Yamaguchi1, J., Nishimura1, Y., Kanada, A., Kobayashi, M., Maemura, Y., Ishida, S., Okano, Y., 2004. Cremophor EL augments the cytotoxicity of hydrogen peroxide in lymphocytes dissociated from rat thymus glands. Toxicol. Lett. 154, 143-148]. The effect of cremophor EL on Ca(2+)-dependent process of cell death has been examined using a flow cytometer since hydrogen peroxide increases intracellular Ca2+ concentration. Cremophor EL at clinically-relevant concentrations greatly increased the population of dead cells in rat thymocytes simultaneously treated with A23187, a calcium ionophore increasing intracellular Ca2+ concentration. Removal of Ca2+ from external solution diminished the cremophor EL-induced increase in the dead cell population. Result suggests that Ca(2+)-dependent process is involved in the cremophor EL-induced decrease in the cell viability in the simultaneous presence of A23187. The population of cells with hypodiploidal DNA was not increased by the application of cremophor EL and A23187 although the cell viability was greatly decreased, indicating that the type of cell death is necrosis. It is suggested that cremophor EL at clinically-relevant concentrations augments the Ca(2+)-dependent process of necrosis.
Article
1. The specifically regulated restrictive permeability barrier to cells and molecules is the most important feature of the blood–brain barrier (BBB). The aim of this review was to summarize permeability data obtained on in vitro BBB models by measurement of transendothelial electrical resistance and by calculation of permeability coefficients for paracellular or transendothelial tracers. 2. Results from primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin are presented. Effects of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the barrier function are discussed. 3. BBB permeability results gained on in vitro models of pathological conditions including hypoxia and reoxygenation, neurodegenerative diseases, or bacterial and viral infections have been reviewed. Effects of cytokines, vasoactive mediators, and other pathogenic factors on barrier integrity are also detailed. 4. Pharmacological treatments modulating intracellular cyclic nucleotide or calcium levels, and activity of protein kinases, protein tyrosine phosphatases, phospholipases, cyclooxygenases, or lipoxygenases able to change BBB integrity are outlined. Barrier regulation by drugs involved in the metabolism of nitric oxide and reactive oxygen species, as well as influence of miscellaneous treatments are also listed and evaluated. 5. Though recent advances resulted in development of improved in vitro BBB model systems to investigate disease modeling, drug screening, and testing vectors targeting the brain, there is a need for checking validity of permeability models and cautious interpretation of data.
Article
The impact of a pharmaceutical relevant metabolizable, ionic surfactant or two synthetic, nonionic surfactants on the absorption and lipoprotein incorporation of a lipophilic drug, retinol, was studied in the Caco-2 cell culture. Filter-grown monolayers of Caco-2 cells were incubated for 20 h with (3)H-retinol and (14)C-oleic acid and with increasing concentrations of lyso-phosphatidylcholine (lyso-PC), Cremophor RH40, or Tween 80. The concentration of (3)H-retinol and (14)C-lipid was measured in the apical, intracellular, and basolateral compartments. The basolateral medium was ultracentrifugated into different lipoprotein classes and their (3)H-retinol and (14)C-lipid concentrations were determined. The cells incubated with lyso-PC and Tween 80 increased the incorporation of (3)H-retinol and (14)C-lipid into chylomicrons and very low density lipoproteins (VLDL). The explored surfactants impacted the incorporation of (3)H-retinol and (14)C-lipid in chylomicrons and VLDL in a concentration-dependent manner. As these surfactants interfere with the intestinal lipoprotein secretion, inclusion of high concentrations of the surfactants in lipid-based formulations of poorly aqueous soluble drugs might impact the degree of intestinal lymphatic transport of the drug after oral administration.