Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways

Polysorbates 20 and 80 (Tween 20 and Tween 80) are used in the formulation of biotherapeutic products for both preventing surface adsorption and as stabilizers against protein aggregation. The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan being polyoxyethylene sorbitan monolaurate for polysorbate 20 and polyoxyethylene sorbitan monooleate for polysorbate 80. The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate. This review focuses on the chemical structure of the polysorbates, factors influencing micelle formation and factors and excipients influencing stability and degradation of the polyoxyethylene and fatty acid ester linkages.     

Contribution of calcium ion sequestration by polyoxyethylated nonionic surfactants to the enhanced colonic absorption of p-aminobenzoic acid

Enhanced absorption of p-aminobenzoic acid (PABA) from the colon by polyoxyethylated nonionic surfactants was investigated using an in situ perfusion technique. The order of their absorption-enhancing effect was as follows: polyoxyethylene lauryl ether greater than polyoxyethylene sorbitan fatty acid esters approximately equal to polyoxyethylene fatty acid esters. The coexistence of calcium chloride in the perfusing solution caused a partial reverse in this enhancement. The calcium ion sequestration capacity of the surfactants was correlated with their ability to enhance colonic absorption of PABA. The findings suggest that calcium ion sequestration by the surfactants contributes to their enhancement of the colonic absorption of PABA.     

Polysorbates as novel lipid-modulating candidates for reducing serum total cholesterol and low-density lipoprotein levels in hyperlipidemic C57BL/6J mice and rats

Polysorbates are amphiphilic, non-ionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan which are widely used in the cosmetic, food and pharmaceutical industries owing to these special characteristics and their low toxicity profiles. In the present study, polysorbates were investigated for their hypolipidemic activity. C57BL/6J mice and Sprague-Dawley rats were fed a high-fat diet for four weeks, then were divided into several groups, normal saline, polysorbates and positive control drugs such as lovastatin and colestyramine were administered orally to the animals for another four weeks. Complete lipid profiles of the experimental animals were determined by assessing the serum levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol. The results indicate that polysorbates significantly lowered the lipid components. Polysorbates are potential candidates for preventing intestinal absorption of redundant lipid from daily intake and subsequently for preventing hyperlipidemia as well as atherosclerosis.     

A simple method for the selection of a suitable emulsifier based on color difference

The purpose of this study was to establish a simple and rapid method for selecting a nonionic surfactant to prepare an emulsion that can maintain a stable emulsification phase. As an index of the degree of emulsification, the white chromaticity of the prepared sample was measured using a color difference meter. When liquid paraffin was used as an oil, the color difference (dE(H)) was shown as a change in a V-shaped curve depending on changes in the hydrophile-lipophile balance (HLB) number of sorbitan ester, polyoxyethylene sorbitan ester, or polyethyleneglycol ester. Although there was a difference in the minimal value of dE(H) in these experiments, the dE(H) was similar to the required HLB of liquid paraffin (HLB=10). On the other hand, no relationship was found between the HLB number and color difference when polyglycerol ester was used. It seems that there was possibility of the effect by the lowering of apparent HLB with a collapse of the hydration. These results indicate that color difference could be utilized for the selection of surfactants, except for polyglycerol ester.     

Design and in vitro characterization of small unilamellar niosomes as ophthalmic carrier of dorzolamide hydrochloride

Abstract

The objective of this work was to formulate and characterize non-ionic surfactant vesicles (niosomes) as an ocular carrier of dorzolamide hydrochloride (Dorzo); one of the antiglaucoma drugs. Niosomes were prepared of Cholesterol (Chol) with sorbitan monoesters (Span 20, 40, 60) or sorbitan trioleate (Span 85) in a molar ratio of 40:150. Those prepared from Span 40 were selected for further investigation on the effect of addition of dicetylphosphate (DCP) and polyoxyethylene fatty acid esters (either Tween 20, 40 or 80). All The batches were prepared using mechanical shaking technique, followed by sonication and then characterized using Zetasizer, transmission electron microscopy (TEM), calculating percent drug entrapment efficiency and cumulative percent released. Z-average sizes of the niosomes were between 25.9 and 165.5?nm. All niosomal formulations showed negative zeta potential charge. Dorzo was successfully entrapped in all of the formulations with entrapment efficiencies ranging between 34.81% and 97.66%. With reference to release profiles, Dorzo-loaded niosomal formulations showed significant reduction in cumulative percent drug released than Dorzo solution. High entrapment efficiencies, biphasic prolonged release rate and small particles size highlight Dorzo-loaded niosomal preparations as a promising ophthalmic carrier to prolong the drug lowering effect on the intraocular pressure.     

Micellar Solubilization of Timobesone Acetate in Aqueous and Aqueous Propylene Glycol Solutions of Nonionic Surfactants

The micellar solubilization of timobesone acetate, a novel topical corticosteroid, was studied in aqueous and aqueous propylene glycol solutions of 1 to 5% nonionic surfactants at 25°C. The surfactants used were polyoxyethylene (POE) sorbitan monofatty acid esters (polysorbates), fatty acid esters (Myrj), and fatty alcohol ethers (Brij), as well as sucrose monolaurate (Crodesta SL40). The increase in the solubility of timobesone acetate in the micellar solutions was dependent on the type and concentration of surfactant. The solubilizing capacity of the surfactant micelles and the distribution coefficient of timobesone acetate in aqueous micellar solutions were found (1) to increase with increasing length of the hydrophobic fatty acid group; (2) to increase according to the structure of the hydrophilic group in the order of POE sorbitan ester, sucrose ester, POE ester, and POE ether; (3) to be unaffected by the increase in POE chain length; and (4) to tend to decrease in surfactant containing unsaturated fatty acid groups. In aqueous propylene glycol solution, the solubilizing capacity increased slightly, i.e., up to 1.5-fold in 50% propylene glycol solution, for the ester-type surfactants (polysorbates and Myrj). But this increase was not observed in the ether-type surfactant (Brij) solution. The distribution coefficient decreased logarithmically with increasing concentrations of propylene glycol in the solution. This was caused by the logarithmic increase in the timobesone acetate solubility in the bulk phase, while the solubility in the micellar phase was practically unchanged. The results support the equilibrium distribution model of micellar solubilization.     

The hydrolysis of indomethacin in aqueous solutions of polysorbates

The effect of surfactants on the rate of hydrolysis of indomethacin is reported for homologous series of polyoxyethylene (20) sorbitan fatty acid esters (polysorbates). The degradation of the solubilized drug follows a first-order process in which the rate decreases with the increasing surfactant concentration. The increasing length of hydrophobic chain of surfactant molecule has little effect on the rate of reaction. The solubilized solutions of indomethacin can be stored for at least one year. The influence of pH on the solubilizing power of polysorbate 80 is also studied.     

Comparison of cytotoxicity of various surfactants tested on normal human fibroblast cultures using the neutral red test, MTT assay and LDH release.

We used the neutral red test, MTT assay and lactate dehydrogenase (LDH) release to compare the potential cytotoxicity of six surfactants belonging to different classes--three non-ionic surfactants (Triton x100, octylphenoxypolyethoxy alcohol, from Orion; Tween 60, polyoxyethylene (20) sorbitan monostearate, from ICI Speciality Chemicals; Tween 80, polyoxyethylene (20) sorbitan monolaurate, from Labosi), two anionic surfactants (Texapon K1298, sodium lauryl sulphate, from Henkel; Texapon N40, sodium laurylether sulphate, from Henkel) and one cationic surfactant (benzethonium chloride, from Siber Hegner)--on human fibroblast cultures. According to the LC50 (microg ml(-1)), the tested surfactants can be classified in the following order of increasing cytotoxicity: Tween 80 < Texapon N40 < Tween 60 < Texapon K1298 < Triton x100 < benzethonium chloride.     

Formulation and physical characterization of water-in-oil microemulsions containing long- versus medium-chain glycerides

Stable self-emulsifying water-in-oil (w/o) microemulsions of extremely small particle size (5–30 nm) and consisting of an oil, a blend of a low and high HLB surfactants and an aqueous phase, have been developed using commercially available and pharmaceutically acceptable components. Their formation was monitored by the corresponding pseudo-ternary phase diagram. The oil phase contained long- or medium-chain triglycerides, and mono-/diglycerides or sorbitan esters (low HLB surfactants). Polysorbate 80 (Tween 80) was used as a high HLB surfactant. Microemulsions were readily prepared by admixing appropriate quantities of the various components with gentle hand-mixing or stirring to ensure thorough mixing. In the case of microemulsions incorporating long-chain glycerides and/or sorbitan esters, high temperature (40–60°C) was used to reduce viscosity and solubilize all components during the formation of microemulsions. Limited levels of aqueous phase (< 10%, w/w) can be solubilized within w/o microemulsions incorporating long-chain glycerides and/or sorbitan esters. Microemulsions containing medium-chain glycerides (mono-/di-/triglycerides) can be formulated at ambient temperature and can solubilize aqueous phase up to 40% (w/w). The conductance, viscosity, refractive index, density and mean particle diameter of a typical w/o microemulsion incorporating medium-chain glycerides (Captex 355/Capmul MCM/Tween 80/saline, 65/22/10/3, % w/w), were: 0.540 μmhos/cm, 56.7 cP, 1.449, 0.9677, and 15.2 ± 4.1 nm (polydispersity of 0.153), respectively. The corresponding values of a w/o microemulsion incorporating long-chain triglycerides and monoglycerides (Soybean oil/Arlacel 186/Tween 80/saline, 65/22/10/3, % w/w) were: 0.177 μmhos/cm, 125.1 cP, 1.471, 0.9010, and 10.3 ± 2.5 nm (polydispersity of 0.114), respectively. Several water-soluble molecules/peptides of different molecular size and charge have been formulated in these w/o microemulsions at pharmacological relevant levels. These systems are discussed in terms of their drug delivery potential.     

Comparison of impact of the different hydrophilic carriers on the properties of piperazine-containing drug.

The objective of this study was to determine the impact of a series of nonionic surfactants on the solubility of piperazine-containing drug (meclizine, MZ) in comparison to that of natural cyclodextrins (alpha-CD and beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD). The solubility of the drug was studied in either CDs solutions or nonionic surfactant solutions. Three classes of nonionic surfactants were used namely; polyoxyethylene (POE) sorbitan fatty acid esters (polysorbates), POE fatty acid esters (Myrjs) and polyethylene oxide (PEO) fatty alcohol ethers (Brijs and Eumulgins). The solubility of MZ was increased linearly with the increasing surfactant concentration, indicating that micellar solubilization follows the partition model. It was found that the longer the hydrocarbon chain in a homologous series, the more efficient is the solubilizing power of surfactant. For example, polysorbate 80 (Tween-80) is a more efficient solubilizer than polysorbate 20 (Tween-20), indicating that the drug was incorporated in the core of micelle more than the capsular region of the micelle. On the other hand, in case of POE fatty acid esters, the solubilizing power increased with decreasing polyoxyethylene chain as Myrj 53 was more efficient than Myrj 59. In class of PEO fatty alcohol ethers, the shorter the hydrophilic chain and longer lipophilic chain, the more efficient was the solubilizing capacity. Thus, Brij 58 was more efficient solubilizer than Brij 35 and Eumulgin C1000 was more active than Eumulgin C1500. Comparatively, Eumulgin C1000 had the highest solubilizing power for MZ among the studied PEO fatty alcohol ethers and other groups of surfactants. The solubility action of surfactants toward MZ was increased by raising the temperature of the surfactant solutions from 30 to 45 degrees C. Hydrophilic macromolecules (PEG 1000 and PEG 6000) or cosolvents (glycerol and propylene glycol) have a very slight effect on the solubility of MZ and confirm the predominance of hydrophobic interaction between the drug and nonionic surfactants. A(L)-type phase solubility diagrams were obtained for the drug with alpha-, beta- and DM-beta-CDs showing that the solubility of MZ was enhanced through inclusion complexation. Comparatively, DM-beta-CD had the highest solubilizing efficiency for the drug among the investigated CDs, which could be attributed to its larger hydrophobic cavity size.     

维生素D2的增溶与稳定性的研究

3种结构类型不同的增溶剂对维生素D₂的增溶效应,聚氧乙烯蓖麻油类大于聚氧乙烯脱水山梨醇脂肪酸酯类及聚氧乙烯脂肪醇醚类。在聚氧乙烯蓖麻油类C-125中维生素D₂的稳定性亦大于其余的两种增溶剂。在吐瘟80胶团中丁基羟基茴香醚主要位于胶团的栅层深部,由于可与维生素D₂处于胶团的相同部位,故较位于栅层浅部及水相之中的没食子酸丙酯,有效地延缓了维生素D₂的自动氧化作用。     

Emulsion stabilization by non-ionic surfactants: the relevance of surfactant cloud point.

The effect of various additives, electrolytes and non-electrolytes, on the cloud point of non-ionic surfactants has been studied. Additives which salt-out the polyoxyethylene chains of the surfactants cause decreased stability of oil-in-water emulsions by decreasing the true hydrophile-lipophile balance (HLB) of the surfactant; additives such as sodium iodide and propanol salt-in the non-ionic surfactants and result in an increase in the effective or true HLB of the system. The latter additives do not increase the hydration of the polyoxyethylene chains but their effect must be on the structure of water so that the heat of hydration of the chains is altered. Experiments with free films of the aqueous surfactant (Brij 96) show that thinning rates are markedly affected by the additives, but there is little effect on the equilibrium thickness of the films (ca 11 nm). Nonetheless the thickness at the transition from thick film to equilibrium black film decreases with increasing cloud point of the solution indicating increased stability. The importance of structure formation in the liquid film separating the emulsion globules was demonstrated.     

Non-ionic surfactants and membrane transport of thioridazine in goldfish.

Polysorbate 80, which has been widely used in studies of the effects of surfactants on drug absorption, increases the rate of absorption of some drugs at concentrations near its critical micelle concentration (cmc). To determine whether all non-ionic surfactants were capable of inducing this effect, the effects of six commercial non-ionic surfactants on thioridazine absorption in goldfish have been compared with the effect of polysorbate 80. The reciprocal death time (T-1) determined when the fish were immersed in the solution under study was the index of absoprtion rate used. Not all surfactants tested increased T-1. Cremophor EL (polyoxyethylated castor oil), Atlas G1295 (a polyoxyethylene fatty glyceride), Atlas G1300 (a polyoxyethylene glyceride ester) had no effect below their cmc's. Those surfactants that did increase T-1 [polysorbate 80 (a polyoxyethylene lanolin derivative), G2162 (a polyoxyethylene oxypropylene monosterate) and Renex 650 (a polyoxyethylene alkyl aryl ether)] display the concentration-dependent behaviour reported previously - a decrease in absorption rate when the surfactant concentration is increased above its cmc. The factor determining whether or not the surfactant will increas absorption rate appears to be the configuration of the surfactant molecule rather than its hydrophile-lipophile balance or its surface activity.     

The Influence of Surface Properties on Uptake of Oil into Complex Coacervate Microcapsules

Abstract— A range of surfactants with different hydrophile-lipophile balance (HLB) values was selected to investigate the influence of interfacial properties on the uptake of oil droplets into complex coacervate microcapsules. The well characterized gelatin/acacia complex coacervate system was used in this study and the encapsulation of squalane, and oleic acid was investigated. The surfactants investigated were Span 85, Span 80, Span 40, egg yolk lecithin, and Tween 80. Combinations of surfactants were utilized to obtain intermediate HLB values. The percentage oil encapsulated was determined gravimetrically, based on the initial concentration and the amount extracted from the microcapsules. The aqueous interfacial tension values of the oils and oil/surfactant systems were measured using the Wilhelmy plate method. The interfacial properties were correlated to the percentage oil uptake by the coacervate phase. The relative hydrophobicity/lipophilicity of the oil influenced its uptake by complex coacervate droplets. The presence of surfactant affected oil uptake, depending on the HLB value of the surfactant or surfactant mixture. Uptake of squalane by the gelatin/acacia coacervates was found to be optimized by the addition of surfactants with HLB values in the range 2·5–6. The percentage uptake of oil decreased rapidly for systems prepared containing surfactants with HLB values outside this range. No correlation was observed between oil uptake by the coacervate phase and the interfacial tension of the oil and oil/surfactant systems with double-distilled deionized water.     

Methotrexate transport from the internal phase of multiple w/o/w emulsions

Release rates of Methotrexate (MTX) encapsulated in the internal phase of w/o/w emulsions stabilized by the interfacial interaction between albumin and sorbitan mono-oleate(Tween 80) were measured as functions of two formulation variables--the oil phase and the secondary emulsifier composition. The release rate was significantly affected by the nature of the oil phase and decreased in the order isopropyl myristate greater than octadecane greater than hexadecane greater than dodecane greater than octane, which was a reflection of the increasing internal droplet size of the emulsions. The release rate data conform with first order kinetics. Comparison of the effective permeability coefficients calculated from the experimental apparent first-order rate constants, with the effective permeability coefficient of water in planar oil layers, containing non-ionic surfactants, determined by a microgravimetric method supported the hypothesis of diffusion of MTX in water loaded inverse micelles. Surfactants with high HLB values, used as the secondary hydrophilic emulsifier increased the release rates, primarily by increasing the rate of diffusion of MTX through the non-aqueous liquid membrane.     

Comparison of oil-in-water emulsions manufactured by microfluidization and homogenization

The purpose of this study was to compare drug-free model submicron oil-in-water (o/w) emulsions manufactured by high-speed homogenization and microfluidization. The study was aimed at evaluating the influence of these two manufacturing processes on the stability of the emulsions with respect to emulsifier concentration. Stability was defined in terms of dispersed droplet diameter growth over time. The study was also directed towards identifying the minimum emulsifier concentrations required by either processing method within the same model o/w systems to produce emulsions viable throughout the study period of three months. The Microfluidizer 110L was found to be more effective than the homogenizer in producing stable o/w submicron emulsions using triglycerides of caprylic/capric acid as the oil phase and combinations of emulsifiers (polyoxyethylene sorbitan oleate with high HLB and sorbitan monooleate with low HLB) at low emulsifier concentrations. Submicron emulsions prepared by the microfluidization process had smaller droplet diameters and exhibited less droplet diameter growth over time compared to high-speed homogenization. At emulsifier concentrations below 20% w/w, the droplet diameter or stability of the dispersed phase of the sub-micron emulsions prepared by either process was found to be dependent on the emulsifier content.     

Solubilizing Excipients in Oral and Injectable Formulations

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.     

Preparation of magnetic gelatin microspheres for the targeting of drugs

Magnetically responsive gelatin microspheres for the targeting of drugs have been prepared using a water-in-oil emulsion technique with chemical cross-linking of the protein. The manufacturing variables affecting microsphere size, size distribution and surface characteristics have been examined as well as the magnetic responsivenessin vitro. Sesame oil was utilized for non-aqueous phase and magnetic gelatin microspheres of different size from 1.89 to 14.88 μm in mean diameter could be obtained with variation of HLB values of non-ionic surfactants. The content of magnetite which uniformly distributed throughout the microspheres was 26.7% (w/w). It was possible to control the localization of magnetic gelatin microspheres at specific sites within capillary models by using external magnetic field of under 5K gauss.     

The effect of the concentration of the water-soluble component on the rheology of some oil-in-water emulsions containing long-chain alcohols

The effect of the concentration of two non-ionic water-soluble components—Sorbester Q12 (polyoxyethylene sorbitan monolaurate) and cetomacrogol 1000—and two ionic water-soluble components—cetrimide and sodium lauryl sulphate—on the rheology of some emulsions has been examined. Emulsions containing oleyl and lauryl alcohol exhibited an increase in consistency with increasing surfactant content. Maximum values for static yield value and apparent viscosity were found to occur with cetostearyl alcohol as the oil-soluble component. The values of the rheological parameters for products containing ionic surfactants tended to be higher than for non-ionics, but molal plots (and preliminary studies with Texofor N4, Texofor FX170 and Solumin FX170SD) suggested that this was an effect due to molecular weight rather than charge.     

Evaluation of penetration enhancement of lidocaine by nonionic surfactants through hairless mouse skin in vitro

The effect of two nonionic surfactants (polyoxyethylene sorbitan monoesters) on percutaneous absorption of lidocaine in the presence of various concentrations of propylene glycol is reported. Comparisons were made in vitro using excised hairless mouse skin as the barrier membrane. Under infinite dose conditions, steady-state flux was enhanced by surfactants at high propylene glycol concentrations. The same trend was observed following application of a thin layer of formulation to the skin (finite-dose conditions). However, penetration behavior was complex due to: (a) changes in vehicle composition following application, (b) temperature changes resulting from evaporation or moisture uptake, and (c) depletion of lidocaine as a result of penetration with compositions that lost water by evaporation. Two peaks in the flux versus time curve were observed. Surfactant monomer concentration in the vehicles was increased in the presence of propylene glycol.