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Nanoparticles (NPs) are increasingly applied in research and development of new therapies. Characterization of NP systems most often include size, shape, size distribution, and charge but information on the chemical stability of NPs and investigation of the presence of dissolved species is most often missing in efficacy studies due to lack of appropriate methods. In this study, a method based on capillary electrophoresis coupled to inductively coupled plasma mass spectrometry (CE-ICP-MS) was established for analysis of selenium (Se) NPs and dissolved Se species in aqueous media. Peak area and migration time precisions (RSD) of 1.4-3.0% and 1.0-2.6%, respectively, were obtained. CE-ICP-MS analysis of a commercially available SeNP suspension (Q-SeNP) revealed large amounts of selenite corresponding to 32% of the total Se content in the suspension, indicating considerable NP degradation upon storage. The CE-ICP-MS method was modified using a coated fused silica capillary in order to analyze SeNPs in human plasma. Peak area and migration time precisions (RSD) in the range of 3.3-10.7% and 0.8-2.8%, respectively, were achieved. Degradation of polyvinyl alcohol (PVA)-coated SeNPs to selenite in human plasma was demonstrated using the modified method. The amounts of SeNP and selenite were estimated based on a correction factor for the ICP-MS signals of PVA-SeNP and dissolved Se. To the best of our knowledge, this is the first study of SeNPs by CE-ICP-MS and highlights the potential of CE-ICP-MS for quantitative characterization of the behavior of SeNPs in biological media.

The initial drug release from in situ forming implants is affected by factors such as the physicochemical properties of the active pharmaceutical ingredient, the type of the excipients utilized, and the surrounding environment. The feasibility of UV-vis imaging for characterization of the initial behavior of poly(d,l-lactide-co-glycolide) (PLGA)/1-methyl-2-pyrrolidinone (NMP) in situ forming implants was investigated. The in vitro release of leuprolide acetate (LA) and implant formation in real time were monitored using dual-wavelength imaging at 280 and 525 nm, respectively, in matrices based on agarose gel and hyaluronic acid (HA) solution emulating the subcutaneous matrix. Three hours upon injection of the pre-formulation, approximately 15% of the total amount of LA administered was found in the agarose gel, while 5% was released from the implant into the HA solution. Concurrently, more extensive swelling of the implants in the HA solution as compared to implants in the agarose gel was observed. Transport of both LA and the solvent NMP was investigated using UV-vis imaging in a small-scale cell where the geometry of the formulation was controlled, showing a linear correlation between drug release and solvent escape. Light microscopy showed that the microstructures of the resulting implants in agarose gel and HA solution were different, which may be attributed to the different solvent exchange rates. UV imaging was also used to examine the interaction of LA with the release medium by characterizing the diffusion of LA in agarose gel, HA solution, and phosphate buffered saline. The reduced LA diffusivity in HA solution as compared to agarose gel and the LA distribution coefficient in the agarose gel-HA system indicated the presence of interactions between LA and HA. Our findings show that the external environment affects the solvent exchange kinetics for in situ forming implants in vitro, resulting in different types of initial release behavior. UV-vis imaging in combination with biorelevant matrices may offer an interesting approach in the development of in situ forming implant delivery systems.

Intra-articular depot injectables based on in situ suspension formation of ester prodrugs of nonsteroidal anti-inflammatory drugs are promising for management of joint pain. As candidates for this delivery approach, 5 diclofenac ester prodrugs comprising different imidazole-containing promoieties were synthesized and their physicochemical properties characterized. In vitro hydrolysis rates were investigated in buffer solutions, in 40% (v/v) human, equine, canine, and rat plasma, and in 80% (v/v) human and equine synovial fluid. Bioconversion of the prodrugs to diclofenac was found to be enzyme-mediated and follow pseudo-first-order kinetics. Large variations in hydrolysis rates were observed between species and between prodrugs, with prodrug half-lives in plasma from canine, rat, horse, and human of 3.44-141 min, 2.51-14 min, 0.58-1.31 min, and 0.23-1.70 min, respectively. Half-lives in human and equine synovial fluid were 1.6- to 28-fold larger than in plasma. The results highlight the significance of species and tissue variation in prodrug design and suggest that the horse may constitute a suitable model for testing the intra-articular depot approach. Two prodrug candidates appeared promising for future in vivo studies based on their rapid in vitro enzyme-mediated bioconversion to diclofenac and physiochemical characteristics.

Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor dispersion analysis (TDA), flow induced dispersion analysis (FIDA), and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of human serum albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully automated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

In the present study, a method for quantitation of the pharmaceutical peptide oxytocin (OT) and its diselenide-containing analogue (SeOT) in human plasma was developed using gradient elution LC-ICP-MS/MS. Plasma samples were precipitated with acetonitrile containing 1.0% TFA in a volume ratio of 1+3 (sample+precipitation agent) before analysis. Post-column isotope dilution analysis (IDA) was applied for quantitation and was compared with external calibration. Both calibration methods appeared to be fit for purpose regarding figures of merit including linearity, precision, LOD, LOQ and recovery. Analysis of OT and SeOT showed that selenium-based analysis is considerably more sensitive and selective compared to the sulfur-based analysis. Despite the relatively simpler setup of external calibration, IDA can be advantageous because it compensates for instrument drift and changes in organic solvent concentration. The method was applied for a stability study showing the degradation of OT and SeOT in plasma. The degradation of SeOT was faster than the degradation of OT in plasma. Thus, possible stability effects should be considered before replacing a disulfide bridge with a diselenide bridge or introducing a diselenide label in a potential drug. Graphical abstract: [Figure not available: see fulltext.]

Buccal films containing a pH modifying excipient may be able to increase bioavailability of drugs with pH-dependent solubility such as saquinavir. Access to suitable in vitro drug release testing methods may facilitate buccal formulation development. This study aimed to explore two release testing methods for characterising buccal films and to elucidate the relationship between microenvironmental pH (pHM, i.e. the pH around the swelling films) and saquinavir release. The Franz diffusion cell method was applicable to investigate the effect of hydroxypropyl methylcellulose (HPMC) grade on saquinavir release. Films containing HPMC K3 LV had a faster saquinavir release than films containing HPMC K100 LV. A UV/Vis imaging method was developed to visualise saquinavir release and pHM changes during the initial dissolution. Within 5 min, the pHM decreased from 6.8 to around 5.4 for HPMC K100 LV-based films containing 11.1 % or 16.6 % (w/w) malic acid. Subsequently, the pHM increased due to increasing concentrations of saquinavir. An increase in malic acid content led to a faster saquinavir release. The combination of methods may be broadly applicable for excipient screening in development of buccal formulations. The imaging approach holds promise for characterizing other pH modifying formulation principles.

The understanding and characterization of protein interactions is crucial for elucidation of complicated biomolecular processes as well as for the development of new biopharmaceutical therapies. Often, protein interactions involve multiple binding, avidity, oligomerization, and are dependent on the local environment. Current analytical methodologies are unable to provide a detailed mechanistic characterization considering all these parameters, since they often rely on surface immobilization, cannot measure under biorelevant conditions, or do not feature a structurally-related readout for indicating formation of multiple bound species. In this work, we report the use of flow induced dispersion analysis (FIDA) for in-solution characterization of complex protein interactions under in vivo like conditions. FIDA is an immobilization-free ligand binding methodology employing Taylor dispersion analysis for measuring the hydrodynamic radius (size) of biomolecular complexes. Here, the FIDA technology is utilized for a size-based characterization of the interaction between TNF-α and adalimumab. We report concentration-dependent complex sizes, binding affinities (Kd), kinetics, and higher order stoichiometries, thus providing essential information on the TNF-α–adalimumab binding mechanism. Furthermore, it is shown that the avidity stabilized complexes involving formation of multiple non-covalent bonds are formed on a longer timescale than the primary complexes formed in a simple 1 to 1 binding event.

In situ forming implants are exposed to an extracellular matrix resembling a gel rather than aqueous solution upon subcutaneous administration. The aim of study was to develop a gel-based release testing system for characterizing the long-term in vitro behavior of in situ forming implants. The gel-based system consisted of an agarose gel mimicking the subcutaneous injection site and a receiver layer comprising phosphate buffer. Poly(D,L-lactide-co-glycolide) in situ forming implants containing leuprolide acetate as the model peptide and N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO) or triacetin as co-solvent were investigated. The gel-based release testing system discriminated between the formulations. Accelerated release data obtained at elevated temperatures were able to predict real-time release applying the Arrhenius equation. Monitoring of the microenvironmental pH of the implants was performed by UV–Vis imaging in the gel-based system at 50 °C. A pH drop (from pH 7.4 to 6.7 for the NMP and DMSO implants, to pH 5.5 for the triacetin implants) within the first day was observed, followed by an increase to pH ∼7.4. The gel-based system coupled with UV imaging offered opportunity for detailed evaluation and prediction of the in vitro performance of long-acting injectables, facilitating future development of in situ depot forming delivery systems.

The purpose of this study was to investigate whole-dosage form UV-vis imaging as a potential tool for functional characterization of excipients used in solid oral dosage forms. To this end, tablets (average mass 260.0 mg, 224.5 mg and 222.1 mg) containing theophylline anhydrate (20 % w/w), 1% (w/w) magnesium stearate, and 79 % (w/w) of either microcrystalline cellulose (MCC, Avicel PH 101) or hydroxypropyl methylcellulose (HPMC, Methocel K15 M or K100 M) were prepared as model systems. Drug liberation from tablets was studied in 0.01 M HCl at 37 degrees C using a Sirius SDi2 equipped with a USP IV type flow cell comprising a UV-vis imaging detector operating at 255 nm and 520 nm. The effluent from the flow cell was passed through a downstream spectrophotometer, and UV-vis spectra in the wavelength range 200-800 nm were recorded every 2 min. The erosion and swelling behavior of the MCC tablets and HPMC K15 M and K100 M tablets were visualized in real time. The swelling of HPMC K15 M and K100 M containing tablets was assessed quantitatively as changes in tablet diameter measured at 520 nm, and was clearly distinguished from the swelling of the MCC tablets. Namely, an increment of 2.5 mm in diameter was determined for the HPMC tablets while the MCC tablets increased by 0.5-1 mm in diameter. Gel layers of variable thickness were observed only for the HPMC K15 M and K100 M tablets. In addition, a relatively high initial liberation rate of theophylline was found for the MCC tablets as compared to the HPMC tablets. UV-vis imaging revealed features of liberation not revealed by simply measuring drug concentration in the dissolution media or by visual assessment. It may be sufficiently sensitive to be further developed for functional characterization of excipients and provide insights into drug-excipient interactions likely to be useful in formulation development. (C) 2020 Elsevier B.V. All rights reserved.

Elucidating the impact of excipient variability on oral product performance in a biopharmaceutical perspective would be beneficial and allow excipient implementation on Quality by Design (QbD) approaches. The current study investigated the impact of varying viscosity of binders (hypromellose (HPMC)) and superdisintegrants (sodium starch glycolate (SSG)) and particle size distribution of lubricants (magnesium stearate (MgSt)) on the in vitro dissolution of a highly and a poorly soluble drug from immediate release formulations. Compendial (pharmacopoeia buffers) and biorelevant (media simulating the gastrointestinal fluids) media and the USP 2 and USP 4 apparatuses were used to assess the exerted excipient effects on drug dissolution. Real-time dissolution UV imaging provided mechanistic insights into disintegration and dissolution of the immediate release formulations. Varying the viscosity type of HPMC or SSG did not significantly affect drug dissolution irrespective of the compound used. Faster drug dissolution was observed when decreasing the particle size of MgSt for the highly soluble drug. The use of real-time dissolution UV Imaging revealed the influential role of excipient variability on tablet disintegration, as for the highly soluble drug, tablets containing high viscosity HPMC or low particle size MgSt disintegrated faster as compared to the control tablets while for the poorly soluble drug, slower tablet disintegration was observed when increasing the viscosity of the HPMC as compared to the control tablets. Changes in drug dissolution when varying excipients may be anticipated if the excipient change has previously affected drug solubility. The use of multivariate data analysis revealed the influential biopharmaceutical factors such as critical excipient types/properties, drug aqueous solubility, medium/hydrodynamic characteristics affecting the impact of excipient variability on in vitro drug dissolution.

Co-amorphous systems comprising low-molecular weight drugs and co-formers constitute an interesting approach to optimize pharmaceutical performance of drugs with low aqueous solubility. Within the different types of co-amorphous systems, the combination of a drug with its own salt may be an attractive formulation option due the absence of any inactive co-formers. The aim of this study was to investigate the possibility of forming a co-amorphous system from naproxen (NAP) and its sodium salt (NAP(Na)). Ball milling of NAP and NAP(Na) at equal molar ratio resulted in the formation of a co-amorphous system whilst NAP and NAP(Na) alone were crystalline following both, ball milling and melt quenching. Infrared spectroscopy and physical stability testing revealed that intermolecular interactions were able to maintain the ball milled NAP-NAP(Na) system amorphous for 2 months at 40 °C. Surprisingly, the dissolution rate of co-amorphous NAP-NAP(Na) was only intermediate between those of crystalline NAP and crystalline NAP(Na). In situ Raman spectroscopic measurements indicated an initial phase separation of the co-amorphous form to NAP and NAP(Na) followed by dissociation of sodium from NAP(Na) and crystallization to NAP. These findings contribute to the design of co-amorphous formulations with the combination of a drug and its own salt.

The study concerns development of simulated synovial fluid for in vitro release testing of drug and prodrug-based depot delivery systems for joint injection. The importance of drug protein binding and esterase-catalyzed prodrug hydrolysis was investigated using a membrane-based in vitro release testing system, the rotating dialysis cell. Protein binding was investigated in synovial fluid from arthritic patients, human plasma and phosphate buffer, pH 7.4, containing serum albumin. Compounds with low (sodium diatrizoate) and high (naproxen and diclofenac) degrees of protein binding were investigated. The cleavage of two poorly soluble naproxen and diclofenac ester prodrugs was studied in esterase-containing media, diluted synovial fluid and plasma. Regarding protein binding, the in vitro release studies revealed that drug release profiles obtained using buffer comprising 2% (w/v) serum albumin, or 40% (v/v) plasma were comparable to profiles obtained employing 80% (v/v) synovial fluid. For prodrug-based depot injectables, application of 40% (v/v) plasma provided release profiles comparable to those using 80% (v/v) synovial fluid. Design of simulated synovial fluids for characterizing in vitro the interplay between drug/prodrug solubility, protein binding and susceptibility to enzymatic cleavage, and joint residence time is feasible. Synovial fluid may be replaced with readily accessible serum albumin solutions or diluted plasma.

For poly (lactide-co-glycolide acid) (PLGA)-based in situ forming implants, the rate of implant formation plays an important role in determining the overall drug release kinetics. Currently, in vitro techniques capable of characterizing the processes of drug release and implant formation at the same time are not available. A hydrogel-based in vitro experimental setup was recently developed requiring only microliter of formulation and forming a closed system potentially suitable for interfacing with various spectroscopic techniques. The aim of the present proof-of-concept study was to investigate the feasibility of concomitant UV imaging, Vis imaging and light microscopy for detailed characterization of the behavior of in situ forming PLGA implants in the hydrogel matrix mimicking the subcutis. The model compounds, piroxicam and α-lactalbumin were added to PLGA-1-methyl-2-pyrrolidinone and PLGA-triacetin solutions. Upon bringing the PLGA-solvent-compound pre-formulation in contact with the hydrogel, Vis imaging and light microscopy were applied to visualize the depot formation and UV imaging was used to quantify drug transport in the hydrogel. As compared to piroxicam, the α-lactalbumin invoked an acceleration of phase separation and an increase of implant size. α-Lactalbumin was released faster from the PLGA-1-methyl-2-pyrrolidinone system than the PLGA-triacetin system opposite to the piroxicam release pattern. A linear relationship between the rate of implant formation and initial compound release within the first 4h was established for the PLGA-NMP systems. This implies that phase separation may be one of the controlling factors in drug release. The rate of implant formation may be an important parameter for predicting and tailoring drug release. The approach combining UV imaging, Vis imaging and light microscopy may facilitate understanding of release processes and holds potential for becoming a useful tool in formulation development of in situ forming implants.

Taylor dispersion analysis (TDA) is an absolute method (no calibration needed) for the determination of the molecular diffusion coefficient (D) based on the band broadening of a solute in a laminar flow. TDA is virtually applicable to any solute with size ranging from angstrom to sub-micrometer. The higher sizing limit is restricted by the occurrence of possibly two regimes: convective and hydrodynamic chromatography (HDC) regimes, which have different physical origins that should not be confused. This work aims at clearly defining the experimental conditions for which these two regimes can play a role, alone or concomitantly. It also calculates the relative error on D due to the HDC regime according to the solute to capillary size ratio. It is demonstrated in this work that HDC does not significantly affect the TDA measurement as long as the hydrodynamic radius of the solute is lower than 0.0051 times the capillary radius. Experimental illustrations of the occurrence of the two regimes are given taking polystyrene nanoparticles as model solutes. Finally, application of TDA to the sizing of large real-life solutes is proposed, taking cubosomes as new drug nanocarriers of potential interest for drug delivery purposes.

Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using a novel UV-vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively, were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of the systems was followed by the study of changes in light transmission and absorbance as a function of time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation was determined and found to decrease with increasing the PLGA concentration from 20% to 40% (w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site environment. The resulting implant morphology depended on the stiffness of hydrogel matrix, indicating that the matrix in which implants are formed is of importance. Overall, the work showed that the UV-vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue holds potential in providing bio-relevant and mechanistic information on the phase separation processes of in situ forming implants.

This work reports a novel approach to the assessment of the surface properties of compacts used in Surface Dissolution Imaging (SDI). SDI is useful for determining intrinsic dissolution rate (IDR), an important parameter in early stage drug development. Surface topography, post-compaction and post-SDI run, have been measured using a non-contact, optical, three-dimensional microscope based on focus variation, the Alicona Infinite Focus Microscope, with the aim of correlating the IDRs to the surface properties. Ibuprofen (IBU) was used as a model poorly-soluble drug. DSC and XRD were used to monitor possible polymorphic changes that may have occurred post-compaction and post-SDI run. IBUs IDR decreased from 0.033 mg/min/cm2 to 0.022 mg/min/cm2 from 10 to 20 min, respectively, during the experiment. XRD and DSC showed no form changes during the SDI run. The surface topography images showed that a distinct imprint was embossed on the surfaces of some compacts which could affect IDRs. Surface parameter values were associated with the SDI experiments which showed strong correlations with the IDR values. The variable-focus microscope can be used as a complimentary tool in the determination of IDR values from the SDI.

New strategies are continuously sought for the treatment of skin and wound infections due to increased problems with non-healing wounds. Electrospun nanofiber mats with antibacterial agents as drug delivery systems provide opportunities for the eradication of bacterial infections as well as wound healing. Antibacterial activities of such mats are directly linked with their drug release behavior. Traditional pharmacopoeial drug release testing settings are not always suitable for analyzing the release behavior of fiber mats intended for the local drug delivery. We tested and compared different drug release model systems for the previously characterized electrospun chloramphenicol (CAM)-loaded nanofiber (polycaprolactone (PCL)) and microfiber (PCL in combination with polyethylene oxide) mats with different drug release profiles. Drug release into buffer solution and hydrogel was investigated and drug concentration was determined using either high-performance liquid chromatography, ultraviolet-visible spectrophotometry, or ultraviolet (UV) imaging. The CAM release and its antibacterial effects in disc diffusion assay were assessed by bacterial bioreporters. All tested model systems enabled to study the drug release from electrospun mats. It was found that the release into buffer solution showed larger differences in the drug release rate between differently designed mats compared to the hydrogel release tests. The UV imaging method provided an insight into the interactions with an agarose hydrogel mimicking wound tissue, thus giving us information about early drug release from the mat. Bacterial bioreporters showed clear correlations between the drug release into gel and antibacterial activity of the electrospun CAM-loaded mats.

The pur­pose of this study was to con­duct an in­ter­lab­o­ra­tory ring-study, with six part­ners (aca­d­e­mic and in­dus­trial), in­ves­ti­gat­ing the mea­sure­ment of in­trin­sic dis­so­lu­tion rate (IDR) us­ing sur­face dis­so­lu­tion imag­ing (SDI) equip­ment. Mea­sure­ment of IDR is im­por­tant in phar­ma­ceu­ti­cal re­search as it pro­vides char­ac­ter­is­ing in­for­ma­tion on drugs and their for­mu­la­tions. This work al­lowed us to as­sess the SDI’s in­ter­lab­o­ra­tory per­for­mance for mea­sur­ing IDR us­ing a de­fined stan­dard op­er­at­ing pro­ce­dure (see sup­port­ing in­for­ma­tion) and six drugs as­signed as low (tadalafil, bromocrip­tine me­sy­late), medium (carvedilol, in­domethacin) and high (ibupro­fen, val­sar­tan) sol­u­bil­ity com­pounds. Fasted State Sim­u­lated In­testi­nal Fluid (FaS­SIF) and blank FaS­SIF (with­out sodium tau­ro­cholate and lecithin) (pH 6.5) were used as me­dia. Us­ing the stan­dard­ised pro­to­col an IDR value was ob­tained for all com­pounds and the re­sults show that the over­all IDR rank or­der matched the sol­u­bil­ity rank or­der. In­ter­lab­o­ra­tory vari­abil­ity was also ex­am­ined and it was ob­served that the vari­abil­ity for lower sol­u­bil­ity com­pounds was higher, co­ef­fi­cient of vari­a­tion >50%, than for in­ter­me­di­ate and high sol­u­bil­ity com­pounds, with the ex­cep­tion of in­domethacin in FaS­SIF medium. In­ter lab­o­ra­tory vari­abil­ity is a use­ful de­scrip­tor for un­der­stand­ing the ro­bust­ness of the pro­to­col and the sys­tem vari­abil­ity. On com­par­i­son to an­other pub­lished small-scale IDR study the rank or­der­ing with re­spect to dis­so­lu­tion rate is iden­ti­cal ex­cept for the high sol­u­bil­ity com­pounds. This re­sults in­di­cates that the SDI ro­bustly mea­sures IDR how­ever, no rec­om­men­da­tion on the use of one small scale method over the other is made.

Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surfac ...

Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations.

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

Buccal delivery of saquinavir has the advantage to bypass the gastrointestinal enzymatic degradation and the hepatic first-pass metabolism. Saquinavir has a pH-dependent solubility and is poorly soluble in human saliva at the physiological pH. Decreasing microenvironmental pH (pH M) in saliva may increase saquinavir release from buccal formulations. The present study aimed to investigate the effects of organic acids on the pH M, saquinavir release in vitro and the solid-state form of saquinavir. An UV/Vis imaging method was used to measure pH M. After 5 min of swelling of the buccal films containing malic acid, pH M was reduced from 6.8 to 5.4. The films containing malic acid were more efficient in maintaining low pH M than films containing citric acid and succinic acid. Addition of organic acids in the buccal films resulted in a faster drug release than films without acids due to the reduced pH M. However, the enhancement of saquinavir release was limited by the fast release of organic acids. Addition of malic acid and citric acid suppressed the crystallization of saquinavir during 3 months storage at the elevated temperature (40 °C) and humidity (RH 75%) respectively. These results suggest that pH M modifying film is a potential formulation strategy for buccal delivery of saquinavir.

There is a need for bio-predictive and well-characterized in vitro release models in the development of intra-articular depot formulations. Here, the commercially-available Scissor system, a membrane-based two-compartment release testing instrument, was applied to characterize the transport and release of the drug diclofenac employing conditions intended to mimic transport in the synovial joint. The fate of hyaluronic acid and human serum albumin, the main bio-relevant components incorporated in the system, was investigated. A promising strategy for providing sustained drug release upon intra-articular administration are lipid-based preformulations forming non-lamellar liquid crystalline phases in situ. The usefulness of the Scissor system for investigating the initial drug release from these delivery systems was evaluated. The diclofenac release rate upon injection of an aqueous solution was influenced by the composition of the injection site matrix, i.e. the hyaluronic acid content. Hyaluronic acid and human serum albumin were found to escape from the donor compartment into the acceptor medium through the employed polycarbonate membrane. Sustained diclofenac release was obtained by formation of highly viscous liquid crystalline phases upon injection of the lipid-based preformulations. The study shows the feasibility and potential of the Scissor system for testing initial release of intra-articular depot formulations of low-molecular-weight drug compounds.

Flow-Induced Dispersion Analysis (FIDA) enables characterization and quantification of proteins under native conditions. FIDA is based on measuring the change in size of a ligand as it selectively interacts with the target protein. The unbound ligand has a relatively small apparent hydrodynamic radius (size), which increase in the presence of the analyte due to binding to the analyte. The Kd of the interaction may be obtained in a titration experiment and the measurement of the apparent ligand size in an unknown sample forms the basis for determining the analyte concentration. The apparent molecular size is measured by Taylor dispersion analysis (TDA) in fused silica capillary capillaries. FIDA is a "ligand-binding" assay and has therefore certain features in common with Enzyme-Linked Immunosorbent Assay (ELISA), Surface Plasmon Resonance (SPR), and Biolayer Interferometry (BLI) based techniques. However, FIDA probes a single in-solution binding event and thus makes assay development straightforward, and the absolute size measurement enables built-in assay quality control. Further, as FIDA does not involve surface chemistries, complications related to nonspecific adsorption of analyte and assay components are minimized enabling direct measurement in, e.g., plasma and serum.

Therapeutic proteins and peptides are mainly administrated by subcutaneous injection. In vitro release testing of subcutaneous injectables performed using methods that take the structure and environment of the subcutaneous tissue into account may improve predictability of the in vivo behavior and thereby facilitate establishment of in vitro in vivo correlations. The aim of the study was to develop a biopredictive flow-through in vitro release method with a gel-type matrix for subcutaneously administered formulations and to explore the possibility of establishing a level A in vitro in vivo correlation for selected insulin products. A novel gel-based flow-through method with the incorporation of an injection step was used to assess selected commercial insulin formulations with different duration of action (Actrapid®, Mixtard® 30, Insulatard®, Lantus®). The in vitro release method provided the correct rank ordering in relation to the in vivo performance. For the modified release insulins Insulatard® and Lantus®, an in vitro in vivo correlation using non-linear time scaling was established based on the in vitro release data and in vivo subcutaneous absorption data of the 125I-labeled insulins taken from literature. Predicted absorption profiles were constructed using the in vitro in vivo correlation and subsequently converted into simulated plasma profiles. The approach taken may be of wider utility in characterizing injectables for subcutaneous administration.

Cisplatin ( cis-diamminedichloroplatinum(II)) is among the most potent cytotoxic agents used in cancer chemotherapy. The encapsulation of cisplatin in lipid-based drug carriers has been challenging owing to its low solubility in both aqueous and lipid phases. Here, we investigated cisplatin encapsulation in nonlamellar liquid-crystalline (LC) nanodispersions formed from a ternary mixture of phytantriol (PHYT), vitamin E (Vit E), and an anionic phospholipid [either phosphatidylglycerol (DSPG) or phosphatidylserine (DPPS)]. We show an increase in cisplatin encapsulation efficiency (EE) in nanodispersions containing 1.5-4 wt % phospholipid. The EE was highest in DPPS-containing nanodispersions (53-98%) compared to DSPG-containing counterparts (25-40%) under similar experimental conditions. Through structural and morphological characterizations involving synchrotron small-angle X-ray scattering and cryogenic transmission electron microscopy, we further show that varying the phospholipid content of cisplatin-free nanodispersions triggers an internal phase transition from a neat hexagonal (H

Evaluation of drug precipitation is important in order to address challenges regarding low and variable bioavailability of poorly water-soluble drugs, to assess potential risk of patient safety with infusion therapy, and to explore injectable in situ suspension-forming drug delivery systems. Generally, drug precipitation is assessed in vitro through solution concentration analysis methods. Dual-wavelength UV-vis imaging is a novel imaging technique that may provide an opportunity for simultaneously monitoring changes in both solution and solid phases during precipitation. In the present study, a multimodal approach integrating UV-vis imaging, light microscopy, and Raman spectroscopy was developed for characterization of piroxicam supersaturation, precipitation, and dissolution in a flow-through setup. A solution of piroxicam dissolved in 1-methyl-2-pyrrolidinone was injected into a flowing aqueous environment (pH 7.4), causing piroxicam to precipitate. Imaging at 405 and 280 nm monitored piroxicam concentration distributions during precipitation and revealed different supersaturation levels dependent on the initial concentration of the piroxicam solution. The combination with imaging at 525 nm, light microscopy, and Raman spectroscopy measurements demonstrated concentration-dependent precipitation and the formation, growth, and dissolution of individual particles. Results emphasize the importance of the specific hydrodynamic conditions on the piroxicam precipitation. The approach used may facilitate comprehensive understanding of drug precipitation and dissolution processes and may be developed further into a basic tool for formulation screening and development.

In the field of drug delivery to the articular cartilage, it is advantageous to apply artificial tissue models as surrogates of cartilage for investigating drug transport and release properties. In this study, artificial cartilage models consisting of 0.5% (w/v) agarose gel containing 0.5% (w/v) chondroitin sulfate or 0.5% (w/v) hyaluronic acid were developed, and their rheological and morphological properties were characterized. UV imaging was utilized to quantify the transport properties of the following four model compounds in the agarose gel and in the developed artificial cartilage models: H-Ala-β-naphthylamide, H-Lys-Lys-β-naphthylamide, lysozyme, and α-lactalbumin. The obtained results showed that the incorporation of the polyelectrolytes chondroitin sulfate or hyaluronic acid into agarose gel induced a significant reduction in the apparent diffusivities of the cationic model compounds as compared to the pure agarose gel. The decrease in apparent diffusivity of the cationic compounds was not caused by a change in the gel structure since a similar reduction in apparent diffusivity was not observed for the net negatively charged protein α-lactalbumin. The apparent diffusivity of the cationic compounds in the negatively charged hydrogels was highly dependent on the ionic strength, pointing out the importance of electrostatic interactions between the diffusant and the polyelectrolytes. Solution based affinity studies between the model compounds and the two investigated polyelectrolytes further confirmed the electrostatic nature of their interactions. The results obtained from the UV imaging diffusion studies are important for understanding the effect of drug physicochemical properties on the transport in articular cartilage. The extracted information may be useful in the development of hydrogels for in vitro release testing having features resembling the articular cartilage.

An analytical method based on capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICP-MS) detection was developed for studies on the interaction of gold-containing drugs and plasma proteins using auranofin as example. A detection limit of 18 ng/mL of auranofin corresponding to 5.2 ng/mL Au and a precision of 1.5 % were obtained. Kinetic studies of the interaction between auranofin and protein were performed by incubation in aqueous solutions as well as 20 % human plasma at 37 °C. The reaction of auranofin with human serum albumin (HSA) and plasma proceeded fast; 50 % of un-bound auranofin disappeared within 2 and 3 min, respectively. By blocking the free cysteine (Cys-34) by iodoacetamide on HSA, it was shown that Cys-34 was the main reaction site for auranofin. By selective labeling of HSA present in 20 % human plasma with iophenoxate, it was demonstrated that HSA was the major auranofin-interacting protein in plasma. The CE-ICP-MS method is proposed as a novel approach for kinetic studies of the interactions between gold-based drugs and plasma proteins. Graphical Abstract Development of a CE-ICP-MS based method allows for studies on interaction of the gold containing drug auranofin with plasma proteins.

Microwave ovens have been used extensively in organic synthesis in order to accelerate reaction rates. Here, a set up comprising a microwave oven combined with silicon carbide (SiC) plates for the controlled microwave heating of model formulations has been applied in order to investigate, if a microwave oven is applicable for accelerated drug stability testing. Chemical interactions were investigated in three selected model formulations of drug and excipients regarding the formation of ester and amide reaction products. In the accelerated stability studies, a design of experiments (DoE) approach was applied in order to be able to rank excipients regarding reactivity: Study A: cetirizine with PEG 400, sorbitol, glycerol and propylene glycol. Study B: 6-aminocaproic acid with citrate, acetate, tartrate and gluconate. Study C: atenolol with citric, tartaric, malic, glutaric, and sorbic acid. The model formulations were representative for oral solutions (co-solvents), parenteral solutions (buffer species) and solid dosage forms (organic acids applicable for solubility enhancement). The DoE studies showed overall that the same impurities were generated by microwave oven heating leading to temperatures between 150°C and 180°C as compared to accelerated stability studies performed at 40°C and 80°C using a conventional oven. Ranking of the reactivity of the excipients could be made in the DoE studies performed at 150-180°C, which was representative for the ranking obtained after storage at 40°C and 80°C. It was possible to reduce the time needed for drug-excipient compatibility testing of the three model formulations from weeks to less than an hour in the three case studies. The microwave oven is therefore considered to be an interesting alternative to conventional thermal techniques for the investigation of drug-excipient interactions during preformulation.

UV imaging provides spatially and temporally resolved absorbance measurements, which are highly useful in pharmaceutical analysis. Commercial UV imaging instrumentation was originally developed as a detector for separation sciences, but the main use is in the area of in vitro dissolution and release testing studies. The review covers the basic principles of the technology and summarizes the main applications in relation to intrinsic dissolution rate determination, excipient compatibility studies and in vitro release characterization of drug substances and vehicles intended for parenteral administration. UV imaging has potential for providing new insights to drug dissolution and release processes in formulation development by real-time monitoring of swelling, precipitation, diffusion and partitioning phenomena. Limitations of current instrumentation are discussed and a perspective to new developments and opportunities given as new instrumentation is emerging.

Human serum albumin (HSA) is the most abundant protein in the human plasma. HSA has several physiological roles in the human body, including storage and transport. Owing to the predominance of albumin in plasma, HSA is often involved in the protein binding of drugs. The aim of this work was to develop a selective, quantitative method for determining albumin in plasma with the purpose of clarifying the fate of metal-based drugs in biological systems. The method can also be applied for determination of urine albumin, which is of relevance in diagnostics of kidney disease. A selective method for quantification of HSA based on labelling the protein with iophenoxic acid (IPA) was developed. Samples were subjected to size exclusion chromatography (SEC) and detection by inductively coupled plasma mass spectrometry (ICP-MS) monitoring iodine and platinum. The iodine signal for the HSA-IPA complex showed linearity in the range 1 to 250 mg L(-1). The precision was 3.7% and the accuracy 100.7% determined by analysis of a certified HSA reference material. The limit of detection (LOD) and limit of quantification (LOQ) were 0.23 and 9.79 mg L(-1), respectively. The method was applied for analysis of HSA in human plasma and urine samples and for studying the binding of cisplatin to proteins in the human plasma.

In the present study, the impact on peptide properties of labelling peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was evaluated. Three differently labelled variants of the cell-penetrating peptide (CPP) penetratin (Pen) were synthesized, PenM(Se), TAMRA-PenM(Se) and TAMRA-Pen. The labelled peptides were characterized in terms of hydrodynamic radius, secondary structure during peptide-membrane interaction, effect on membrane leakage induction, uptake efficiency in HeLa cells. Furthermore, stability of peptides and identities of degradation products in cell media and cell lysate were evaluated. TAMRA-labelling increased the hydrodynamic radius of Pen and PenM(Se) significantly. Labelling with Se caused no or minimal changes in size, secondary structure and membrane leakage induction in concentration levels relevant for cellular uptake studies. Similar degradation patterns of all labelled peptides were observed in HBSS media; degradation was mainly due to oxidation. Cellular uptake was significantly higher for the TAMRA labelled peptides as compared to Se-labelled Pen. Extensive degradation was observed in media during cellular uptake studies, however, in all cell lysates, primarily the intact peptide (PenM(Se), TAMRA-PenM(Se) or TAMRA-Pen) was observed. Selenium labelling caused minimal alteration of the physicochemical properties of the peptide and allowed for absolute quantitative determination of cellular uptake by inductively coupled plasma mass spectrometry. Selenium is thus proposed as a promising alternative label for quantification of peptides in general, altering the properties of the peptide to a minor extent as compared to commonly used peptide labels.

Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system. In the presence of the analyte the apparent diffusivity of the indicator changes due to complexation. This change in diffusivity is used to quantify the analyte. This approach, termed Flow Induced Dispersion Analysis (FIDA), is characterized by being fast (minutes), selective (quantification is possible in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator-analyte dissociation constant which in favourable cases is in the sub-nanomolar to picomolar range for antibody-antigen interactions.

Peptides are usually administered through subcutaneous injection. For low potency drugs, this may require high concentration formulations increasing the risk of peptide aggregation, especially for compounds without any intrinsic chargeable groups. Carbetocin was used as a model to study the behavior of uncharged peptides at high concentrations. Manipulation of the aggregation behavior of 70 mg/mL carbetocin was attempted by selecting excipients which interact with hydrophobic groups in carbetocin, and cover hydrophobic surfaces and interfaces. Peptide aggregation was induced by shaking stress and followed over time. Carbetocin solutions showed significant visible particle formation already after 4 h of shaking stress. This particle formation was not due to supersaturation or phase separation but suggested a nucleated aggregation process. None of the excipients prevented carbetocin aggregation, though altered aggregation behavior was observed, such as induction of fibril formation for most, but not all, charged excipients. Sodium dodecyl sulfate was found to accelerate peptide aggregation both below and above the critical micelle concentration in half-filled vials. However, in the absence of an air headspace, sodium dodecyl sulfate above the critical micelle concentration was capable of preventing shaking-induced carbetocin aggregation. Our study highlights the complexity in rational excipient selection to stabilize uncharged peptides at high concentration.

Long-acting nonsteroidal anti-inflammatory drug formulations for intra-articular injection might be effective in the management of joint pain and inflammation associated sports injuries and osteoarthritis. In this study, a prodrug-based delivery system was evaluated. The synthesized diclofenac ester prodrug, a weak base (pKa 7.52), has relatively high solubility at low pH (6.5 mg mL(-1) at pH 4) and much lower solubility at physiological pH (4.5 μg mL(-1) at pH 7.4) at 37°C. In biological media including 80% (v/v) human synovial fluid (SF), the prodrug was cleaved to diclofenac mediated by esterases. In situ precipitation of the prodrug was observed upon addition of a concentrated slightly acidic prodrug solution to phosphate buffer or SF at pH 7.4. The degree of supersaturation accompanying the precipitation process was more pronounced in SF than in phosphate buffer. In the rotating dialysis cell model, a slightly acidic prodrug solution was added to the donor cell containing 80% SF resulting in a continuous appearance of diclofenac in the acceptor phase for more than 43 h after an initial lag period of 8 h. Detectable amounts of prodrug were found in the rat joint up to 8 days after knee injection of the acidic prodrug solution.

Variable dissolution from sodium salts of drugs containing a carboxylic acid group after passing the acidic environment of the stomach may affect oral bioavailability. The aim of the present proof of concept study was to investigate pH effects in relation to the dissolution of sodium naproxenate in 0.01M hydrochloric acid. For this purpose a UV/vis imaging-based approach capable of measuring microenvironmental pH in the vicinity of the solid drug compact as well as monitoring drug dissolution was developed. Using a pH indicating dye real-time spatially resolved measurement of pH was achieved. Sodium naproxenate, can significantly alter the local pH of the dissolution medium, is eventually neutralized and precipitates as the acidic species naproxen. The developed approach is considered useful for detailed studies of pH dependent dissolution phenomena in dissolution testing.

Poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamines (DSPE-mPEGs) are a family of amphiphilic lipopolymers attractive in formulating injectable long-circulating nanoparticulate drug formulations. In addition to long circulating liposomes, there is an interest in developing injectable long-circulating drug nanocarriers based on cubosomes and hexosomes by shielding and coating the dispersed particles enveloping well-defined internal nonlamellar liquid crystalline nanostructures with hydrophilic PEG segments. The present study attempts to shed light on the possible PEGylation of these lipidic nonlamellar liquid crystalline particles by using DSPE-mPEGs with three different block lengths of the hydrophilic PEG segment. The effects of lipid composition, PEG chain length, and temperature on the morphology and internal nanostructure of these self-assembled lipidic aqueous dispersions based on phytantriol (PHYT) were investigated by means of synchrotron small-angle X-ray scattering and Transmission Electron Cryo-Microscopy. The results suggest that the used lipopolymers are incorporated into the water-PHYT interfacial area and induce a significant effect on the internal nanostructures of the dispersed submicrometer-sized particles. The hydrophilic domains of the internal liquid crystalline nanostructures of these aqueous dispersions are functionalized, i.e., the hydrophilic nanochannels of the internal cubic Pn3m and Im3m phases are significantly enlarged in the presence of relatively small amounts of the used DSPE-mPEGs. It is evident that the partial replacement of PHYT by these PEGylated lipids could be an attractive approach for the surface modification of cubosomal and hexosomal particles. These PEGylated nanocarriers are particularly attractive in designing injectable cubosomal and hexosomal nanocarriers for loading drugs and/or imaging probes.

Drug formulations containing polyethylene glycol may give rise to formation of reaction products between the aforementioned and the active pharmaceutical ingredient. Supercritical fluid chromatography has recently achieved new interest and improved instrumentation is now available. Here, supercritical fluid chromatography has been evaluated for its possible use for determination of reactions products formed between polyethylene glycol and active pharmaceutical ingredients. A mixture of polyethylene glycols with average molecular weights of 400-6000Da was separated with supercritical fluid chromatography using silica columns and carbon dioxide modified with methanol as mobile phase. Satisfactory resolution (Rs=1.2) of the individual oligomers up to a molecular weight of 1000Da was obtained using evaporative light scattering as detection technique. The active pharmaceutical ingredients, cetirizine or indomethacin were investigated in a reaction mixture containing polyethylene glycol 400 after incubation at 80°C for 120h. Polyethylene glycol esters formed upon reaction with both active pharmaceutical ingredients were observed as polymeric patterns with ultraviolet detection and identified with mass spectrometry. Cetirizine was observed to be more reactive than indomethacin. The observed difference in reactivity is due to differences in polar and steric effects between cetirizine and indomethacin. Evaporative light scattering, ultraviolet absorbance and mass spectrometric detection were investigated and each detection technique has its own advantages and disadvantages, but in order to be able to detect selected impurities in the complex mixture of impurities formed, mass spectrometry is superior.

The purpose of this study was to investigate the in vitro release of 5-aminosalicylic acid from single extrudates by UV imaging and to explore the technique as a visualization tool for detecting film coating defects on extrudates coated with a thin ethyl cellulose layer. 5-Aminosalicylic acid extrudates were film coated with ethyl cellulose in a typical lab system coater equipped with one Wurster partition. Dissolution testing was performed first in a conventional paddle dissolution apparatus and second, in a flow through geometry equipped with a UV imaging system. Selected film coated extrudates from four different coating levels were placed in agarose gels and UV imaging was performed for a total of 240 min. Absorbance maps were obtained thus visualizing the release of 5-aminosalicylic acid over time and it was possible to detect a decrease in release as a function of increased ethyl cellulose coating weight gain. Using a calibration curve the released amount was calculated and the individual release profiles for each coating weight gain in general resulted in comparable release profiles. Furthermore, the release profiles were consistent with the dissolution results obtained from the paddle dissolution testing. The release from defect extrudates was visualized by the absorbance maps and the release was highest from the compromised part of the extrudates. UV imaging has proven to be a useful technique to visualize the release of 5-aminosalicylic acid from single film coated extrudates and it has potential for detection of film coating defects.

Intra-articular injection of oil solutions of lipophilic prodrugs that rapidly degrade to their parent compound in synovial fluid may constitute a feasible approach to increase the joint residence time of non-steroidal anti-inflammatory drugs. In this in vivo study, oil solutions of the N,N-diethyl glycolamide ester prodrug of naproxen (16 mg/ml) were injected into the rat knee joint by dosing 6 μl formulation per 100 g body weight. The sustained release properties were compared to those of intra-articularly injected aqueous and oil solutions of naproxen by monitoring the naproxen serum concentrations over time. Two oils, medium-chain triglycerides and castor oil, differing with respect to viscosity were tested. After intra-articular administration of oil prodrug solutions, a significant increase in the time to maximum naproxen serum concentration from around 40 to 245 min, an increase in the MRTj from around 0.11 to 3.3 h and a 30% reduction in the maximum serum concentration were observed compared to that of the parent naproxen. The similar serum profiles obtained using the two oils indicate that the release was not affected by the oil viscosity. A prolonged naproxen joint residence time in rats was shown by intra-articular injection of an oil prodrug solution.

The aim of the present work was to investigate the interaction between bile salts present in the intestine of man, dog and rat with the negatively charged cyclodextrin (CD), sulfobutylether-β-cyclodextrin (SBEβCD). The interactions between bile salts and CDs are of importance for the release of CD-complexed drugs upon oral administration. This makes a good understanding of this particular interaction important for rational drug formulation. SBEβCD is a modified CD, which has attracted particular interest in formulation science. It is unique in the sense that it carries approximately seven negatively charged side chains, which can potentially interact electrostatically with the guest molecule. Bile salts are negatively charged at physiological pH, and the concomitant repulsion from SBEβCD could potentially reduce their affinity for this CD and hence their ability to expel drugs delivered as SBEβCD complexes. However, this study has demonstrated that the interaction, between a bile salts and SBEβCD is only slightly weaker than the corresponding interactions with natural βCD. Significant differences between the thermodynamics of bile salt complexes with respectively HPβCD and SBEβCD were found, when comparing the same degree of substitution. This underscores the importance of the substituents on the interactions of modified CDs with bile salts.

Interactions of drug candidates with the biomacromolecules of the synovial fluid affect drug targeting to the articular cartilage as well as clearance from the synovial space upon intra-articular administration. Hyaluronic acid (HA) and human serum albumin (HSA) are two main components existing in the synovial fluid. To this end, we investigated the affinity of seven cationic amino acid and dipeptide β-naphthylamide derivatives towards HA and HSA in order to shed light on possible relationships between physicochemical properties, in particular charge state, and biomacromolecular interactions to increase the joint residence time. Capillary electrophoresis frontal analysis was used for characterization of the binding of the derivatives to hyaluronic acid and HSA at 25 °C in acetate buffer (pH 4.65) and phosphate buffer (pH 7.40), respectively. Linear binding isotherms were observed for the ligand–hyaluronic acid interactions and the obtained binding constants ranged from 43 to 133 M−1. The average fraction of bound ligand towards hyaluronic acid increased with increasing the net charge of the ligands but was less than 67 % for all investigated ligands. The obtained binding constants of the ligands with HSA varied in the range of 103–106 M−1. The interactions of low-molecular weight derivatives with hyaluronic acid were highly dependent on the ligand charge state. This trend was not observed for the interactions with HSA. The obtained affinity data may provide useful information in the design of cartilage adhesive prodrugs with extended residence time in the synovial cavity.

Self-Nanoemulsifying Drug Delivery Systems (SNEDDSs) were developed using well-defined excipients with the objective of mimicking digested SNEDDSs without the use of enzymes and in vitro lipolysis models and thereby enabling studies of the morphology and size of nanoemulsions as well as digested nanoemulsions by Cryo-TEM imaging and Dynamic Light Scattering. Four SNEDDSs (I-IV) were developed. Going from SNEDDS I to IV lipid content and solubility of the model drug cinnarizine decreased, which was also the case for dispersion time and droplet size. Droplet size of all SNEDDS was evaluated at 1% (w/w) dispersion under different conditions. Cinnarizine incorporation increased the droplet size of SNEDDSs I and II whereas for SNEDDSs III and IV no difference was observed. At low pH cinnarizine had no effect on droplet size, probably due to increased aqueous solubility and partitioning into the aqueous phase. Dispersion of the SNEDDSs in Simulated Intestinal Media (SIM) containing bile salts and phospholipids resulted in a decrease in droplet size for all SNEDDS, as compared to dispersion in buffer. Increasing the bile salt/phospholipid content in the SIM decreased the droplet sizes further. Mimicked digested SNEDDS with highest lipid content (I and II) formed smaller nanoemulsion droplet sizes upon dispersion in SIM, whereas droplet size from III and IV were virtually unchanged by digestion. Increasing the bile acid/phosphatidylcholine content in the SIM generally decreased droplet size, due to the solubilizing power of the endogenous surfactants. Digestion of SNEDDSs II resulted in formation of vesicles or micelles in fasted and fed state SIM, respectively. The developed and characterized SNEDDS provide for a better knowledge of the colloid phases generated during digestion of SNEDDS and therefore will enable studies that may yield a more detailed understanding of SNEDDS performance.

Upon subcutaneous administration, the distribution of drug between the delivery vehicle and the biological tissue critically affects the absorption of drug substances. Utilization of physical models resembling the native tissues appears promising for obtaining a detailed understanding of the performance of drug delivery systems based on in vitro experiments. The objective of this study was to evaluate a UV imaging-based method for real-time characterization of the release and transport of piroxicam in hydrogel-based subcutaneous tissue mimics/surrogates. Piroxicam partitioning from medium chain triglyceride (MCT) into 0.5% (w/v) agarose or 25% (w/v) F127-based hydrogels was investigated by monitoring the concentration profiles of the drug in the gels. The effect of pH on piroxicam distribution and diffusion coefficients was studied. For both hydrogel systems, the diffusion of piroxicam in the gels was not affected significantly by the pH change from 4.0 to 7.4 but a considerable change in the oil-gel distribution coefficients was found (24 and 34 times less at pH 7.4 as compared those observed at pH 4.0 for F127 and agarose gels, respectively). In addition, the release and transport processes of piroxicam upon the injection of aqueous or MCT solutions into an agarose-based hydrogel were investigated by UV imaging. The spatial distribution of piroxicam around the injection site in the gel matrix was monitored in real-time. The disappearance profiles of piroxicam from the injected aqueous solution were obtained. This study shows that the UV imaging methodology has considerable potential for characterizing transport properties in hydrogels, including monitoring the real-time spatial concentration distribution in vitro after administration by injection.

Liposomes are self-assembled phospholipid vesicles and have numerous research and therapeutic applications. In the pharmaceutical and biomedical sciences liposomes find use as models of biological membranes, partitioning medium and as drug carriers. The present review addresses the use of capillary electrophoresis and liposome electrokinetic chromatography for the characterization of liposomes in a pharmaceutical context. Capillary electrophoretic techniques have been used for the measurement of electrophoretic mobility, which provides information on liposome surface charge, size and membrane permeability of liposomes. The use of liposome electrokinetic chromatography and capillary electrophoresis for determination of liposome/water partitioning and characterization of drug-liposome interactions is reviewed. A number of studies indicate that capillary electrophoresis may have a role in the characterization of liposome drug delivery systems, e.g., for the investigation of encapsulation efficiency and drug leakage. The well-known characteristics of capillary electrophoresis, i.e., low sample volume requirement, high separation efficiency in aqueous media without a stationary phase, minimal sample preparation, and a high degree of automation, makes it an attractive approach in liposome research.

A capillary electrophoresis inductively coupled plasma mass spectrometry method for separation of free cisplatin from liposome-encapsulated cisplatin and protein-bound cisplatin was developed. A liposomal formulation of cisplatin based on PEGylated liposomes was used as model drug formulation. The effect of human plasma matrix on the analysis of liposome-encapsulated cisplatin and intact cisplatin was studied. The presence of 1 % of dextran and 4 mM of sodium dodecyl sulfate in HEPES buffer was demonstrated to be effective in improving the separation of liposomes and cisplatin bound to proteins in plasma. A detection limit of 41 ng/mL of platinum and a precision of 2.1 % (for 10 μg/mL of cisplatin standard) were obtained. Simultaneous measurements of phosphorous and platinum allows the simultaneous monitoring of the liposomes, liposome-encapsulated cisplatin, free cisplatin and cisplatin bound to plasma constituents in plasma samples. It was demonstrated that this approach is suitable for studies of the stability of liposome formulations as leakage of active drug from the liposomes and subsequent binding to biomolecules in plasma can be monitored. This methodology has not been reported before and will improve characterization of liposomal drugs during drug development and in studies on kinetics.

Sulfobutyl ether-β-cyclodextrin (SBEβCD) is utilized in preformulation and drug formulation as an excipient for solubilization of drugs with poor aqueous solubility. Approximately seven negative charges of SBEβCD play a role with respect to solubilization and complexation, but also have an influence on the ionic strength of the background electrolyte when the cyclodextrin is used in capillary electrophoresis. Mobility-shift affinity capillary methods for investigation of the complexation of taurocholate and taurochenodeoxycholate with the negatively charged cyclodextrin derivative applying constant power and ionic strength conditions as well as constant voltage and varying ionic strength were investigated. A new approach for the correction of background electrolyte ionic strength was developed. Mobility-shift affinity capillary electrophoresis experiments obtained at constant voltage and constant power settings were compared and found to provide binding parameters that were in good agreement upon correction. The complexation of taurochenodeoxycholate with SBEβCD was significantly stronger than the corresponding interaction involving taurocholate. The obtained stability constants for the bile salts were in the same range as those previously reported for the interaction with neutral β-cyclodextrins derivatives, i.e. the positions of the negative charges on SBEβCD and the bile salts within the complex did not lead to significant electrostatic repulsion.

A novel real-time UV imaging approach for non-intrusive investigation of the diffusion and partitioning phenomena occurring during piroxicam release from medium chain triglyceride (MCT) solution into two hydrogel matrices is described. Two binary polymer/buffer gel matrices, 0.5% (w/v) agarose and 25% (w/v) Pluronic F127, were applied as simple models mimicking the subcutaneous tissue. The evolution of the absorbance maps as a function of time provided detailed information on the piroxicam release processes upon the exposure of the gel matrices to MCT. Using calibration curves, the concentration maps of piroxicam in the UV imaging area were determined. Regression of the longitudinal concentration-distance profiles, which were obtained using expressions derived from Fick's second law, provided the diffusivity and the distribution coefficients of piroxicam penetrated into the gels. The obtained MCT-agarose (pH 7.4) distribution coefficient of 1.4 was identical to the MCT-aqueous (pH 7.4) distribution coefficient determined by the shake-flask method whereas that of the MCT-Pluronic F127 system was four times less. The experimental data show that UV imaging may have considerable potential for investigating the transport properties of drug formulations intended for the subcutaneous administration.