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  • 1.
    Dahlgren, David
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Biopharmaceutical aspects of intestinal drug absorption: Regional permeability and absorption-modifying excipients2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Before an orally administered drug reaches the systemic circulation, it has to dissolve in the intestinal fluids, permeate across the intestinal epithelial cell barrier, and pass through the liver. The permeation rate of drug compounds can be low and show regional differences.

    The thesis had two general aims. The first of these was, to determine and compare regional intestinal permeability values of model compounds in human and dog. The second was to understand the possible effects of absorption-modifying pharmaceutical excipients (AMEs) on the intestinal permeability of the model compounds. The usefulness of several preclinical animal models for predicting the impact of regional intestinal permeability and AMEs in human was also investigated.

    There was a good correlation between human and dog permeability values in the small intestines for the model compounds. The colon in dog was substantially more permeable than the human colon to the low permeability drug, atenolol. This difference in colonic permeability may have implications for the use of dog as a model species for prediction of human intestinal drug absorption.

    There were no effects of AMEs on the intestinal permeability of any of the high permeability compounds, in any animal model. In the rat single-pass intestinal perfusion model, there was a substantial increase in permeability of all low permeability drugs, induced by two AMEs, chitosan and SDS. This AME-induced increase was substantially lower in the more in vivo relevant rat and dog intraintestinal bolus models. A shorter AME exposure-time in the rat single-pass intestinal perfusion model (15 vs. 75 min) could, however, predict the result from the bolus studies in rat and dog. This illustrates the impact of intestinal transit and mucosal exposure time on AME effects in vivo. The intestinal luminal conditions and enteric neural activity also had an impact on determinations of drug permeability in the rat single-pass intestinal perfusion model, which can have implications for its in vivo relevance.

    In summary, this thesis used multiple in vivo models to evaluate the impact of several biopharmaceutical processes on intestinal drug absorption. This has led to an increased understanding of these absorption mechanisms.

    Delarbeten
    1. Regional Intestinal Permeability of Three Model Drugs in Human
    Öppna denna publikation i ny flik eller fönster >>Regional Intestinal Permeability of Three Model Drugs in Human
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    2016 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 13, nr 9, s. 3013-3021Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Currently there are only a limited number of determinations of human P-eff in the distal small intestine and none in the large intestine. This has hindered the validation of preclinical models with regard to absorption in the distal parts of the intestinal tract, which can be substantial for BCS class II-IV drugs, and drugs formulated into modified-release (MR) dosage forms. To meet this demand, three model drugs (atenolol, metoprolol, and ketoprofen) were dosed in solution intravenously, and into the jejunum, ileum, and colon of 14 healthy volunteers. The P-eff of each model drug was then calculated using a validated deconvolution method. The median P-eff of atenolol in the jejunum, ileum, and colon was 0.45, 0.15, and 0.013 X 10(-4) cm/s, respectively. The corresponding values for metoprolol were 1.72, 0.72, and 1.30 X 10(-4) cm/s, and for ketoprofen 8.85, 6.53, and 3.37 X 10(-4) cm/s, respectively. This is the first study where the human Peff of model drugs has been determined in all parts of the human intestinal tract in the same subjects. The jejunal values were similar to directly determined values using intestinal single-pass perfusion, indicating that the deconvolution method is a valid approach for determining regional P-eff. The values from this study will be highly useful in the validation of preclinical regional absorption models and in silico tools.

    Nyckelord
    intestinal permeability, regional intestinal drug absorption, effective permeability, pharmacokinetics
    Nationell ämneskategori
    Farmaceutiska vetenskaper Gastroenterologi
    Identifikatorer
    urn:nbn:se:uu:diva-307864 (URN)10.1021/acs.molpharmaceut.6b00514 (DOI)000382713700016 ()27504798 (PubMedID)
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, FP7/2007-013
    Tillgänglig från: 2016-11-22 Skapad: 2016-11-22 Senast uppdaterad: 2018-10-20Bibliografiskt granskad
    2. Regional Intestinal Permeability in Dogs: Biopharmaceutical Aspects for Development of Oral Modified-Release Dosage Forms
    Öppna denna publikation i ny flik eller fönster >>Regional Intestinal Permeability in Dogs: Biopharmaceutical Aspects for Development of Oral Modified-Release Dosage Forms
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    2016 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 13, nr 9, s. 3022-3033Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The development of oral modified-release (MR) dosage forms requires an active pharmaceutical ingredient (API) with a sufficiently high absorption rate in both the small and large intestine. Dogs are commonly used in preclinical evaluation of regional intestinal absorption and in the development of novel MR dosage forms. This study determined regional intestinal effective permeability (P-eff) in dogs with the aim to improve regional Peff prediction in humans. Four model drugs, atenolol, enalaprilat, metoprolol, and ketoprofen, were intravenously and regionally dosed twice as a solution into the proximal small intestine (P-SI) and large intestine (LI) of three dogs with intestinal stomas. Based on plasma data from two separate study occasions for each dog, regional Peff values were calculated using a validated intestinal deconvolution method. The determined mean P-eff values were 0.62, 0.14, 1.06, and 3.66 X 10(-4) cm/s in the P-SI, and 0.13, 0.02, 1.03, and 2.20 X 10(-4) cm/s in the LI, for atenolol, enalaprilat, metoprolol, and ketoprofen, respectively. The determined P-SI Peff values in dog were highly correlated (R-2 = 0.98) to the historically directly determined human jejunal P-eff after a single-pass perfusion. The determined dog P-SI P-eff values were also successfully implemented in GI-Sim to predict the risk for overestimation of LI absorption of low permeability drugs. We conclude that the dog intestinal stoma model is a useful preclinical tool for determination of regional intestinal permeability. Still, further studies are recommended to evaluate additional APIs, sources of variability, and formulation types, for more accurate determination of the dog model in the drug development process.

    Nyckelord
    dog intestinal permeability, regional intestinal drug absorption, bioavailability, effective permeability, pharmacokinetics, intestinal perfusion, pharmaceutical development
    Nationell ämneskategori
    Veterinärmedicin Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-307865 (URN)10.1021/acs.molpharmaceut.6b00515 (DOI)000382713700017 ()27500599 (PubMedID)
    Forskningsfinansiär
    EU, Europeiska forskningsrådet, FP7/2007-013
    Tillgänglig från: 2016-11-22 Skapad: 2016-11-22 Senast uppdaterad: 2018-09-12Bibliografiskt granskad
    3. Preclinical Effect of Absorption Modifying Excipients on Rat Intestinal Transport of Model Compounds and the Mucosal Barrier Marker 51Cr-EDTA
    Öppna denna publikation i ny flik eller fönster >>Preclinical Effect of Absorption Modifying Excipients on Rat Intestinal Transport of Model Compounds and the Mucosal Barrier Marker 51Cr-EDTA
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    2017 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 14, nr 12, s. 4243-4251Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    There is a renewed interest from the pharmaceutical field to develop oral formulations of compounds, such as peptides, oligonucleotides, and polar drugs. However, these often suffer from insufficient absorption across the intestinal mucosal barrier. One approach to circumvent this problem is the use of absorption modifying excipient(s) (AME). This study determined the absorption enhancing effect of four AMEs (sodium dodecyl sulfate, caprate, chitosan, N-acetylcysteine) on five model compounds in a rat jejunal perfusion model. The aim was to correlate the model compound absorption to the blood-to-lumen clearance of the mucosal marker for barrier integrity, 51Cr-EDTA. Sodium dodecyl sulfate and chitosan increased the absorption of the low permeation compounds but had no effect on the high permeation compound, ketoprofen. Caprate and N-acetylcysteine did not affect the absorption of any of the model compounds. The increase in absorption of the model compounds was highly correlated to an increased blood-to-lumen clearance of 51Cr-EDTA, independent of the AME. Thus, 51Cr-EDTA could be used as a general, sensitive, and validated marker molecule for absorption enhancement when developing novel formulations.

    Nyckelord
    absorption modifiers, bioequivalence, intestinal perfusion, permeation enhancers, pharmaceutical development
    Nationell ämneskategori
    Fysiologi Farmakologi och toxikologi
    Identifikatorer
    urn:nbn:se:uu:diva-343167 (URN)10.1021/acs.molpharmaceut.7b00353 (DOI)000417342400013 ()28737406 (PubMedID)
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, FP7/2007-013
    Tillgänglig från: 2018-02-26 Skapad: 2018-02-26 Senast uppdaterad: 2018-09-12Bibliografiskt granskad
    4. The effects of three absorption-modifying critical excipients on the in vivo intestinal absorption of six model compounds in rats and dogs.
    Öppna denna publikation i ny flik eller fönster >>The effects of three absorption-modifying critical excipients on the in vivo intestinal absorption of six model compounds in rats and dogs.
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    2018 (Engelska)Ingår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 547, nr 1-2, s. 158-168Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Pharmaceutical excipients that may affect gastrointestinal (GI) drug absorption are called critical pharmaceutical excipients, or absorption-modifying excipients (AMEs) if they act by altering the integrity of the intestinal epithelial cell membrane. Some of these excipients increase intestinal permeability, and subsequently the absorption and bioavailability of the drug. This could have implications for both the assessment of bioequivalence and the efficacy of the absorption-enhancing drug delivery system. The absorption-enhancing effects of AMEs with different mechanisms (chitosan, sodium caprate, sodium dodecyl sulfate (SDS)) have previously been evaluated in the rat single-pass intestinal perfusion (SPIP) model. However, it remains unclear whether these SPIP data are predictive in a more in vivo like model. The same excipients were in this study evaluated in rat and dog intraintestinal bolus models. SDS and chitosan did exert an absorption-enhancing effect in both bolus models, but the effect was substantially lower than those observed in the rat SPIP model. This illustrates the complexity of the AME effects, and indicates that additional GI physiological factors need to be considered in their evaluation. We therefore recommend that AME evaluations obtained in transit-independent, preclinical permeability models (e.g. Ussing, SPIP) should be verified in animal models better able to predict in vivo relevant GI effects, at multiple excipient concentrations.

    Nyckelord
    Absorption modifiers, Bioequivalence, Intraintestinal administration, Permeation enhancers, Pharmaceutical development
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Biofarmaci
    Identifikatorer
    urn:nbn:se:uu:diva-358463 (URN)10.1016/j.ijpharm.2018.05.029 (DOI)000439096500016 ()29758344 (PubMedID)
    Tillgänglig från: 2018-08-29 Skapad: 2018-08-29 Senast uppdaterad: 2018-09-28Bibliografiskt granskad
    5. Effect of absorption-modifying excipients, hypotonicity, and enteric neural activity in an in vivo model for small intestinal transport.
    Öppna denna publikation i ny flik eller fönster >>Effect of absorption-modifying excipients, hypotonicity, and enteric neural activity in an in vivo model for small intestinal transport.
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    2018 (Engelska)Ingår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 549, nr 1-2, s. 239-248, artikel-id S0378-5173(18)30532-5Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The small intestine mucosal barrier is physiologically regulated by the luminal conditions, where intestinal factors, such as diet and luminal tonicity, can affect mucosal permeability. The intestinal barrier may also be affected by absorption-modifying excipients (AME) in oral drug delivery systems. Currently, there is a gap in the understanding of how AMEs interact with the physiological regulation of intestinal electrolyte transport and fluid flux, and epithelial permeability. Therefore, the objective of this single-pass perfusion study in rat was to investigate the effect of three AMEs on the intestinal mucosal permeability at different luminal tonicities (100, 170, and 290 mOsm). The effect was also evaluated following luminal administration of a nicotinic receptor antagonist, mecamylamine, and after intravenous administration of a COX-2 inhibitor, parecoxib, both of which affect the enteric neural activity involved in physiological regulation of intestinal functions. The effect was evaluated by changes in intestinal lumen-to-blood transport of six model compounds, and blood-to-lumen clearance of 51Cr-EDTA (a mucosal barrier marker). Luminal hypotonicity alone increased the intestinal epithelial transport of 51Cr-EDTA. This effect was potentiated by two AMEs (SDS and caprate) and by parecoxib, while it was reduced by mecamylamine. Consequently, the impact of enteric neural activity and luminal conditions may affect nonclinical determinations of intestinal permeability. In vivo predictions based on animal intestinal perfusion models can be improved by considering these effects. The in vivo relevance can be increased by treating rats with a COX-2 inhibitor prior to surgery. This decreases the risk of surgery-induced ileus, which may affect the physiological regulation of mucosal permeability.

    Nyckelord
    Absorption-modifying excipients, Biopharmaceutical classification system, In vivo predictions, Intestinal perfusion, Intestinal permeability, Intestinal physiology, Permeation enhancers
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Biofarmaci
    Identifikatorer
    urn:nbn:se:uu:diva-358464 (URN)10.1016/j.ijpharm.2018.07.057 (DOI)000443255300022 ()30055302 (PubMedID)
    Tillgänglig från: 2018-08-29 Skapad: 2018-08-29 Senast uppdaterad: 2018-11-15Bibliografiskt granskad
    6. Time-dependent effects on small intestinal transport by absorption-modifying excipients.
    Öppna denna publikation i ny flik eller fönster >>Time-dependent effects on small intestinal transport by absorption-modifying excipients.
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    2018 (Engelska)Ingår i: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, artikel-id S0939-6411(18)30804-XArtikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The relevance of the rat single-pass intestinal perfusion model for investigating in vivo time-dependent effects of absorption-modifying excipients (AMEs) is not fully established. Therefore, the dynamic effect and recovery of the intestinal mucosa was evaluated based on the lumen-to-blood flux (Jabs) of six model compounds, and the blood-to-lumen clearance of 51Cr-EDTA (CLCr), during and after 15- and 60-min mucosal exposure of the AMEs, sodium dodecyl sulfate (SDS) and chitosan, in separate experiments. The contribution of enteric neurons on the effect of SDS and chitosan was also evaluated by luminal coadministration of the nicotinic receptor antagonist, mecamylamine. The increases in Jabs and CLCr (maximum and total) during the perfusion experiments were dependent on exposure time (15 and 60 min), and the concentration of SDS, but not chitosan. The increases in Jabs and CLCr following the 15-min intestinal exposure of both SDS and chitosan were greater than those reported from an in vivo rat intraintestinal bolus model. However, the effect in the bolus model could be predicted from the increase of Jabs at the end of the 15-min exposure period, where a six-fold increase in Jabs was required for a corresponding effect in the in vivo bolus model. This illustrates that a rapid and robust effect of the AME is crucial to increase the in vivo intestinal absorption rate before the yet unabsorbed drug in lumen has been transported distally in the intestine. Further, the recovery of the intestinal mucosa was complete following 15-min exposures of SDS and chitosan, but it only recovered 50% after the 60-min intestinal exposures. Our study also showed that the luminal exposure of AMEs affected the absorptive model drug transport more than the excretion of 51Cr-EDTA, as Jabs for the drugs was more sensitive than CLCr at detecting dynamic mucosal AME effects, such as response rate and recovery. Finally, there appears to be no nicotinergic neural contribution to the absorption-enhancing effect of SDS and chitosan, as luminal administration of 0.1 mM mecamylamine had no effect. .........

    Nyckelord
    Biopharmaceutical Classification System, absorption modifiers, bioequivalence, epithelial recovery, intestinal perfusion, intestinal permeability, permeation enhancers, pharmaceutical development
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-359823 (URN)10.1016/j.ejpb.2018.09.001 (DOI)30179738 (PubMedID)
    Tillgänglig från: 2018-09-06 Skapad: 2018-09-06 Senast uppdaterad: 2018-09-12
  • 2.
    Jonsson, Henrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Confined Compression of Single Particles: Development of a Novel Triaxial Testing Instrument and Particle-Scale Modelling2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    When predicting the performance of a powder compaction process, assessing the behaviour of the particles comprising the powder bed is of central relevance. Currently, however, no experimental methods are available for mimicking the multiaxial loading conditions imposed on the individual particles in a powder bed during compaction, and such analyses are therefore usually performed in silico. Thus, the purpose of this thesis is to introduce a novel experimental method that enables experimental evaluation of confined triaxial loading conditions on individual particles in the mm-scale.

    The work underlying the thesis consists of three major parts. Firstly, the triaxial instrument was designed and developed, after which its performance was evaluated using nominally ideal elastic-plastic spheres as model materials. These initial experiments showed that the instrument was able to successfully impose confined triaxial conditions on the particles, something that was verified by finite element method (FEM) simulations.

    Secondly, the triaxial instrument was used to investigate differences in deformation characteristics under uniaxial and triaxial loading conditions for four different microcrystalline cellulose (MCC)-based granules. It was shown that fragmentation, associated with unconfined uniaxial compression, was impeded under confined triaxial conditions, despite the emergence of cracks. In addition, it was observed that the primary crack always occurs in a plane parallel to the most deformed direction, and that the location of the largest pore has a pronounced influence on the path of the crack.

    Thirdly, the influence of different triaxial loading ratios were evaluated on polymer spheres, after which a unified description of contact pressure development was devised. Data from these experiments were then successfully used to calibrate a contact model for simulating bulk powder compression with the discrete element method (DEM).

    All in all, a novel experimental method has been established, which has proven useful as an alternative and complement to numerical studies when studying single particle deformation under confined triaxial conditions.

    Delarbeten
    1. An apparatus for confined triaxial testing of single particles
    Öppna denna publikation i ny flik eller fönster >>An apparatus for confined triaxial testing of single particles
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    2015 (Engelska)Ingår i: Powder Technology, ISSN 0032-5910, E-ISSN 1873-328X, Vol. 270, s. 121-127Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    A novel triaxial apparatus employing overlapping rigid boundaries has been designed and constructed for experimental measurement of contact forces under confined compression of single granules in the mm-scale. The performance of the apparatus was evaluated by performing uniaxial and triaxial compression experiments on ideal elastic-plastic materials. Compression curves were compared with the fully plastic Abbott-Firestone contact model and with results from FEM simulations. The increase in contact force associated with confined conditions was observed in the compression curves from triaxial compression experiments, as supported by predictions from simulations using single particle contact models. Hence, a new method for the assessment of mechanical behaviour of single particles under confined compression can be considered as established.

    Nyckelord
    Compression, Triaxial, Single particles, Confined conditions, Apparatus design, Contact mechanics
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-239203 (URN)10.1016/j.powtec.2014.10.016 (DOI)000347579300014 ()
    Forskningsfinansiär
    Vetenskapsrådet, 621-2011-4049
    Tillgänglig från: 2014-12-19 Skapad: 2014-12-19 Senast uppdaterad: 2018-04-12Bibliografiskt granskad
    2. Investigations of single microcrystalline cellulose-based granules subjected to confined triaxial compression
    Öppna denna publikation i ny flik eller fönster >>Investigations of single microcrystalline cellulose-based granules subjected to confined triaxial compression
    2016 (Engelska)Ingår i: Powder Technology, ISSN 0032-5910, E-ISSN 1873-328X, Vol. 289, s. 79-87Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Confined triaxial compression of single granules was performed in order to assess the contact force development and modes of granule deformation under these conditions. In the study, four microcrystalline cellulose-based granule types of different characteristics were investigated. Results from triaxial single-granule compression experiments were evaluated using an analytical model as well as by comparison to unconfined single-granule compression and to confined bulk compression experiments. It was observed that single granules deform and densify, but tend to keep their integrity during confined triaxial compression, as evident from both compression data and from morphological analysis. Results from confined single granule compression were well represented by the analytical model. These results also largely reflected those from bulk compression experiments, including features of the force-displacement curves as well as rank order between the granule types in terms of contact stiffness. Furthermore, it was shown that intragranular porosity to a high extent governs the onset of plastic incompressibility.

    Nyckelord
    Compression, Triaxial, Hydrostatic, Single granules, Confined conditions, Contact mechanics
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-280895 (URN)10.1016/j.powtec.2015.11.051 (DOI)000370095400012 ()
    Forskningsfinansiär
    Vetenskapsrådet, 621-2011-4049
    Tillgänglig från: 2016-03-16 Skapad: 2016-03-16 Senast uppdaterad: 2018-04-12Bibliografiskt granskad
    3. Mechanical behaviour of ideal elastic-plastic particles subjected to different triaxial loading conditions
    Öppna denna publikation i ny flik eller fönster >>Mechanical behaviour of ideal elastic-plastic particles subjected to different triaxial loading conditions
    2017 (Engelska)Ingår i: Powder Technology, ISSN 0032-5910, E-ISSN 1873-328X, Vol. 315, s. 347-355Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The contact force development for two types of polymeric elastoplastic particles subjected to different triaxial loading conditions was studied experimentally utilising a unique triaxial testing apparatus. In order to evaluate the experimental results, a finite element analysis was performed. The experimental findings highlighted the importance of contact dependence, which manifested itself in two principally different ways. Firstly, a reduced stiffness was observed when plastic deformation ceased to be fully contained, which, depending on the loading conditions, occurred at an engineering strain of about 5-10%. Secondly, a markedly increased stiffness was observed when particle confinement inhibited further plastic deformation, making elastic volume reduction the predominant deformation mode. The experimental results could be well reproduced by the numerical simulations, provided that isotropic hardening was included in the elastoplastic model. In an attempt to invariantly describe the data, a nominal contact pressure was determined as a function of the volumetric constraint of the particle. This resulted in an adequate collapse of results obtained for different loading conditions onto a single master curve at large volumetric constraint. In summary, this paper should be considered as a step along the pathway towards our long term goal of introducing novel and improved contact models.

    Nyckelord
    Particle mechanics, Triaxial, Compression, Spatial confinement, Contact dependence
    Nationell ämneskategori
    Teknisk mekanik
    Identifikatorer
    urn:nbn:se:uu:diva-323755 (URN)10.1016/j.powtec.2017.04.005 (DOI)000401593600041 ()
    Forskningsfinansiär
    Vetenskapsrådet, 621-2011-4049
    Tillgänglig från: 2017-06-13 Skapad: 2017-06-13 Senast uppdaterad: 2018-04-12Bibliografiskt granskad
    4. Crack nucleation and propagation in microcrystalline-cellulose based granules subject to uniaxial and triaxial load
    Öppna denna publikation i ny flik eller fönster >>Crack nucleation and propagation in microcrystalline-cellulose based granules subject to uniaxial and triaxial load
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Cracking patterns of four kinds of granules, based on the common pharmaceutical excipient microcrystalline cellulose (MCC) and subject to compressive load, were examined. The initial pore structure and the location of initial failure under uniaxial compression were assessed using X-ray micro-computed tomography, whereas contact force development and onset of cracking under more complex compressive load were examined using a triaxial testing apparatus. Smoothed particle hydrodynamics (SPH) simulations were employed for numerical analysis of the stress distributions prior to cracking. For granules subject to uniaxial compression, initial cracking always occurred along the meridian and the precise location of the crack depended on the pore structure. Likewise, for granules subject to triaxial compression, the fracture plane of the primary crack was generally parallel to the dominant loading direction. The occurrence of cracking was highly dependent on the triaxiality ratio, i.e. the ratio between the punch displacements in the secondary and dominant loading directions. Compressive stresses in the lateral directions, induced by triaxial compression, prevented crack opening and fragmentation of the granule, something that could be verified in the SPH simulations. These results provide corroboration as well as further insights into previously observed differences between confined and unconfined compression of granular media.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Galenisk farmaci
    Identifikatorer
    urn:nbn:se:uu:diva-347457 (URN)
    Tillgänglig från: 2018-04-03 Skapad: 2018-04-03 Senast uppdaterad: 2018-04-12
    5. Evaluation of bulk compression using a discrete element procedure calibrated with data from triaxial experiments on single particles
    Öppna denna publikation i ny flik eller fönster >>Evaluation of bulk compression using a discrete element procedure calibrated with data from triaxial experiments on single particles
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Confined compression of bimodal mixtures of ideal spherical cellulose acetate (CA) particles (diameters 1.5 and 2.0 mm) was studied numerically with the discrete element method (DEM) and experimentally using a materials tester equipped with suitable tablet tooling. An extended truncated sphere contact model was used in the simulations, enabling them to be carried out to high relative densities (approaching and sometimes exceeding unity). In order to calibrate this model, the contact pressure development was extracted from prior experimental investigations on single 2.0-mm large CA particles. Results from the simulations were evaluated with the Kawakita and Heckel compression equations and compared to the corresponding data obtained from bulk compression experiments. Generally, a high degree of similarity between experiments and simulations was observed, showing the usefulness of combining confined single particle compression experiments with a suitable numerical model when predicting the performance of powder compression to high relative densities.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-347458 (URN)
    Tillgänglig från: 2018-04-03 Skapad: 2018-04-03 Senast uppdaterad: 2018-04-12
  • 3.
    Roos, Carl
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Intestinal absorption of drugs: The impact of regional permeability, nanoparticles, and absorption-modifying excipients2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    For successful delivery of orally given drug products, the drug compounds must have adequate solubility and permeability in the human gastrointestinal tract. The permeability of a compound is determined by its size and lipophilicity, and is usually evaluated in various pre-clinical models, including rat models.

    This thesis had three major aims: 1) investigate regional permeability in human and rat intestines and evaluate two different rat models, 2) investigate the mechanisms behind absorption in nanosuspensions, and 3) investigate the effect of food on the absorption of drug molecules in solutions and suspensions, and also food’s effect on absorption modifying excipients (AMEs).

    Effective human permeability values obtained using regional intra-intestinal dosing and a deconvolution method agreed with values established by perfusion from the jejunum, demonstrating the accuracy and validity of the intra-intestinal bolus-dosing approach. Single-pass intestinal perfusion (SPIP) in rats showed better correlation with human effective permeability than the Ussing chamber, and was therefore deemed the better model for predicting drug permeability in humans.

    Absorption of microsuspensions and nanosuspension was investigated using rat SPIP, which showed that microsuspensions are subject to pronounced food effects, probably by partitioning of drug into the colloidal structures formed by bile acids, lecithin, and fatty acids. Nanosuspensions were less affected by food, which was attributed to fewer available nanoparticles in the fed state due to partitioning into colloidal structures, and because nanoparticles are able to cross the aqueous boundary layer on their own, increasing the concentration of drug adjacent to the epithelial membrane.

    AMEs had less effect in the fed state than the fasted state when investigated using SPIP. This difference may be caused by AMEs partitioning into luminal colloidal structures, decreasing the AMEs’ effects on the intestinal membrane. It thus seems that AMEs as well as drug compounds are subject to food-drug interactions, which may either increase or decrease the effect or absorption, something that needs to be considered during development of new drug products. 

    In summary, this thesis has improved the knowledge of pre-clinical absorption models and the understanding of several biopharmaceutical mechanisms important for drug absorption.

    Delarbeten
    1. Regional Intestinal Permeability of Three Model Drugs in Human
    Öppna denna publikation i ny flik eller fönster >>Regional Intestinal Permeability of Three Model Drugs in Human
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    2016 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 13, nr 9, s. 3013-3021Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Currently there are only a limited number of determinations of human P-eff in the distal small intestine and none in the large intestine. This has hindered the validation of preclinical models with regard to absorption in the distal parts of the intestinal tract, which can be substantial for BCS class II-IV drugs, and drugs formulated into modified-release (MR) dosage forms. To meet this demand, three model drugs (atenolol, metoprolol, and ketoprofen) were dosed in solution intravenously, and into the jejunum, ileum, and colon of 14 healthy volunteers. The P-eff of each model drug was then calculated using a validated deconvolution method. The median P-eff of atenolol in the jejunum, ileum, and colon was 0.45, 0.15, and 0.013 X 10(-4) cm/s, respectively. The corresponding values for metoprolol were 1.72, 0.72, and 1.30 X 10(-4) cm/s, and for ketoprofen 8.85, 6.53, and 3.37 X 10(-4) cm/s, respectively. This is the first study where the human Peff of model drugs has been determined in all parts of the human intestinal tract in the same subjects. The jejunal values were similar to directly determined values using intestinal single-pass perfusion, indicating that the deconvolution method is a valid approach for determining regional P-eff. The values from this study will be highly useful in the validation of preclinical regional absorption models and in silico tools.

    Nyckelord
    intestinal permeability, regional intestinal drug absorption, effective permeability, pharmacokinetics
    Nationell ämneskategori
    Farmaceutiska vetenskaper Gastroenterologi
    Identifikatorer
    urn:nbn:se:uu:diva-307864 (URN)10.1021/acs.molpharmaceut.6b00514 (DOI)000382713700016 ()27504798 (PubMedID)
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, FP7/2007-013
    Tillgänglig från: 2016-11-22 Skapad: 2016-11-22 Senast uppdaterad: 2018-10-20Bibliografiskt granskad
    2. Regional Intestinal Permeability in Rats: A Comparison of Methods
    Öppna denna publikation i ny flik eller fönster >>Regional Intestinal Permeability in Rats: A Comparison of Methods
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    2017 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 14, nr 12, s. 4252-4261Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Currently, the screening of new drug candidates for intestinal permeation is typically based on in vitro models which give no information regarding regional differences along the gut. When evaluation of intestinal permeability by region is undertaken, two preclinical rat models are commonly used, the Ussing chamber method and single-pass intestinal perfusion (SPIP). To investigate the robustness of in vivo predictions of human intestinal permeability, a set of four model compounds was systematically investigated in both these models, using tissue specimens and segments from the jejunum, ileum, and colon of rats from the same genetic strain. The influence of luminal pH was also determined at two pH levels. Ketoprofen had high and enalaprilat had low effective (P-eff) and apparent (P-app) permeability in all three regions and at both pH levels. Metoprolol had high P-eff in all regions and at both pHs and high P-app at both pHs and in all regions except the jejunum, where P-app was low. Atenolol had low P-eff in all regions and at both pHs, but had high P-app at pH 6.5 and low P-app at pH 7.4. There were good correlations between these rat in situ P-eff (SPIP) and human in vivo P-eff determined previously for the same compounds by both intestinal perfusion of the jejunum and regional intestinal dosing. The results of this study indicate that both investigated models are suitable for determining the regional permeability of the intestine; however, the SPIP model seems to be the more robust and accurate regional permeability model.

    Nyckelord
    intestinal permeability, Ussing chamber method, single-pass intestinal perfusion, jejunum, ileum, colon, rat
    Nationell ämneskategori
    Farmakologi och toxikologi
    Identifikatorer
    urn:nbn:se:uu:diva-345176 (URN)10.1021/acs.molpharmaceut.7b00279 (DOI)000417342400014 ()28920690 (PubMedID)
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, FP7/2007-013
    Tillgänglig från: 2018-03-08 Skapad: 2018-03-08 Senast uppdaterad: 2018-10-20Bibliografiskt granskad
    3. In Vivo Mechanisms of Intestinal Drug Absorption from Aprepitant Nanoformulations
    Öppna denna publikation i ny flik eller fönster >>In Vivo Mechanisms of Intestinal Drug Absorption from Aprepitant Nanoformulations
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    2017 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 14, nr 12, s. 4233-4242Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Over recent decades there has been an increase in the proportion of BCS class II and IV drug candidates in industrial drug development. To overcome the biopharmaceutical challenges associated with the less favorable properties of solubility and/or intestinal permeation of these substances, the development of formulations containing nanosuspensions of the drugs has been suggested. The intestinal absorption of aprepitant from two nanosuspensions (20 mu M and 200 mu M total concentrations) in phosphate buffer, one nanosuspension (200 mu M) in fasted-state simulated intestinal fluid (FaSSIF), and one solution (20 mu M) in FaSSIF was investigated in the rat single-pass intestinal perfusion model. The disappearance flux from the lumen (J(disapp)) was faster for formulations containing a total concentration of aprepitant of 200 mu M than for those containing 20 mu M, but was unaffected by the presence of vesicles. The flux into the systemic circulation (J(app)) and, subsequently, the effective diffusion constant (D-eff) were calculated using the plasma concentrations. J(app) was, like J(disapp), faster for the formulations containing higher total concentrations of aprepitant, but was also faster for those containing vesicles (ratios of 2 and 1.5). This suggests that aprepitant is retained in the lumen when presented as nanoparticles in the absence of vesicles. In conclusion, increased numbers of nanoparticles and the presence of vesicles increased the rate of transport and availability of aprepitant in plasma. This effect can be attributed to an increased rate of mass transport through the aqueous boundary layer (ABL) adjacent to the gut wall.

    Nyckelord
    intestinal drug absorption, aprepitant nanoformulations, nanosuspensions, fasted-state simulated intestinal fluid, aqueous boundary layer
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-345174 (URN)10.1021/acs.molpharmaceut.7b00294 (DOI)000417342400012 ()28737398 (PubMedID)
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, FP7/2007-013
    Tillgänglig från: 2018-03-08 Skapad: 2018-03-08 Senast uppdaterad: 2018-10-20Bibliografiskt granskad
    4. Jejunal absorption of aprepitant from nanosuspensions: Role of particle size, prandial state and mucus layer.
    Öppna denna publikation i ny flik eller fönster >>Jejunal absorption of aprepitant from nanosuspensions: Role of particle size, prandial state and mucus layer.
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    2018 (Engelska)Ingår i: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 132, s. 222-230, artikel-id S0939-6411(18)30760-4Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The number of highly lipophilic active pharmaceutical ingredients (APIs) in pharmaceutical development has been constantly increasing over recent decades. These APIs often have inherent issues with solubility and dissolution, limiting their oral bioavailability. Traditionally, a reduction in particle size to the micrometer range has been used to improve dissolution. More recently, size reduction to the nanometer range has been introduced, which further increases the dissolution rate, but may also involve other mechanisms for increasing bioavailability. The effect of particle size on the absorption of aprepitant was investigated using the single-pass intestinal perfusion (SPIP) model in the rat jejunum. Phosphate buffer, fasted-state simulated intestinal fluid (FaSSIF), and fed-state simulated intestinal fluid (FeSSIF) were used as perfusion media to increase understanding of the processes involved and the effects of colloidal structures. The role of mucus on intestinal absorption was investigated by adding the mucolytic agent N-acetyl-cysteine (NAC). The absorption of aprepitant from the nanosuspensions was similar with all perfusion media (buffer = FaSSIF = FeSSIF), whereas food had a pronounced effect on absorption from the microsuspensions (FeSSIF > FaSSIF > buffer). The colloidal structures hence contributed to absorption from the microsuspensions. Partitioning of aprepitant from the nanosuspension into the colloidal structures decreased the amount of nanoparticles available, which offset the effect of food. The appearance flux of aprepitant in blood was non-significantly decreased for nanosuspensions of aprepitant with NAC versus without NAC in buffer (ratio of 2:1), indicating that particle deposition in the mucus may have been decreased as the layer thinned, with subsequently reduced intestinal absorption. The study also showed that the SPIP model is suitable for investigating detailed absorption mechanisms using complex perfusion media, which increase the biorelevance of the model.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-363157 (URN)10.1016/j.ejpb.2018.09.022 (DOI)30266667 (PubMedID)
    Tillgänglig från: 2018-10-15 Skapad: 2018-10-15 Senast uppdaterad: 2018-10-20
    5. Effects of absorption-modifying excipients on jejunal drug absorption in simulated fasted and fed luminal conditions
    Öppna denna publikation i ny flik eller fönster >>Effects of absorption-modifying excipients on jejunal drug absorption in simulated fasted and fed luminal conditions
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    (Engelska)Ingår i: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441Artikel i tidskrift (Refereegranskat) Submitted
    Abstract [en]

    The pharmaceutical industry, prescribers, and patients have all traditionally preferred oral administration of drug products. In recent years there has been an increase in drug candidates with low solubility and/or low permeability, which may limit the use of oral administration. To increase the possibility of oral administration for the poorly permeating drugs, the use of absorption modifying excipients (AMEs) has been proposed, with the aim of increasing the fraction of dose absorbed. The effects of AMEs have previously been investigated in various animal models, including the single-pass intestinal perfusion (SPIP) in rats. To further improve the biorelevance and the in vivo predictiveness of the SPIP model, four compounds (atenolol, enalaprilat, ketoprofen, metoprolol) were perfused in fasted or fed state simulated intestinal fluid (FaSSIF or FeSSIF) together with the AMEs N-acetyl-cysteine, caprate, or sodium dodecyl sulphate. For the poorly permeating compounds enalaprilat and atenolol, the flux was increased the most by the addition of SDS in both FaSSIF and FeSSIF. For ketoprofen, the flux decreased in the presence of all AMEs in at least one of the perfusion media. The flux of metoprolol was not affected by any of the excipients. The changes in magnitude in the compounds’ absorptions were in general smaller in FeSSIF than in FaSSIF, possibly due to differences in colloidal structures present in FeSSIF that made the AMEs less available. The results in FeSSIF were similar to those from bolus-dosing in rat, which further suggests that the effect of AMEs on permeability is strongly affected by interactions between AMEs and colloidal structures in the intestinal lumen. The results suggest that, when investigating the effects of AMEs, the biorelevance of the SPIP method can be increased by the addition of intraluminal constituents to the perfusate.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-363192 (URN)
    Tillgänglig från: 2018-10-20 Skapad: 2018-10-20 Senast uppdaterad: 2018-10-20
  • 4.
    Nyström, Lina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Microgels as Carriers for Antimicrobial Peptides: Surface-bound microgels, and factors affecting peptide interactions2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    With a growing number of multi-resistant bacteria against conventional antibiotics, there is an urgent need to identify new antimicrobial therapeutics. One example that has gained considerable interest is antimicrobial peptides (AMPs). For AMPs to reach their full potential as therapeutics, as well as for other peptide and protein drugs, the right drug delivery system may overcome reported shortcomings, such as fast clearance in the bloodstream and proteolytic degradation. Microgels are weakly cross-linked polymer colloids, which can be made responsive to various stimuli. In the context of drug delivery, microgels are of particular interest as carriers for biomacromolecular drugs, such as peptides and proteins, as their water-rich environment offers both protection against enzymatic degradation and triggered release possibilities. Combining these, the aim of this thesis was to investigate electrostatically triggered surface-bound microgels as a delivery system for AMPs, as well as evaluate such systems as an antimicrobial and anti-inflammatory coating for biomaterials.

    Results presented in this thesis demonstrate effects of microgel charge density, pH, and ionic strength on microgel volume transitions at solid interfaces, surface-induced microgel deformation and nanomechanical properties. In addition, effects of both microgel properties (charge density) and peptide properties (molecular weight, charge density, and posttranslational modifications) on peptide loading and release from surface-bound microgels were investigated. The presented thesis also reports in vitro studies of AMP-loaded microgels in dispersion and surface-bound, as either mono- or multilayers. Notably, the interplay between surface- and release-related effects for the antimicrobial properties of AMP-loaded microgels are investigated. In addition, anti-inflammatory properties of AMP-loaded microgels are also reported.

    Taken together, microgels prove an interesting and versatile drug delivery system for AMPs. Results obtained in this thesis have demonstrated that several key factors need to be taken into consideration in the development of surface-bound microgels as a carrier for AMPs, and that small changes in microgel and peptide properties can alter peptide loading and release profiles.

    Delarbeten
    1. Electrostatic Swelling Transitions in Surface-Bound Microgels
    Öppna denna publikation i ny flik eller fönster >>Electrostatic Swelling Transitions in Surface-Bound Microgels
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    2016 (Engelska)Ingår i: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 8, nr 40, s. 27129-27139Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Herein, electrostatic swelling transitions of poly (ethyl acrylate-co-methacrylic acid) microgels covalently bound to silica surfaces are investigated. Confined at a solid surface, microgel swelling is anisotropically hindered and the structure is flattened to an extent dictated by pH and microgel composition. Microgel deformation under applied load is also shown to depend on microgel charge density, with the highest deformation observed at intermediate charge densities. Two modes of microgel deformation under load were observed, one elastic and one viscoelastic, related to polymer strand deformation and displacement of trapped water, respectively. Results on polymer strand dynamics reveal that the microgels are highly dynamic, as the number of strand-tip interaction points increases 4-fold during a 10 s contact time. Furthermore, finite element modeling captures these effects qualitatively and shows that stress propagation in the microgel network decays locally at the rim of contact with a solid interface or close to the tip probe. Taken together, the results demonstrate a delicate interplay between the surface and microgel which determines the structure and nanomechanical properties of the latter and needs to be controlled in applications of systems such as pH-responsive surface coatings in biomaterials.

    Nyckelord
    atomic force microscopy, finite element method, microgel, pH-responsive, quartz crystal microbalance, surface-bound
    Nationell ämneskategori
    Biomaterialvetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-307540 (URN)10.1021/acsami.6b09751 (DOI)000385469000081 ()27644921 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet, 2012-1842 2013-4384Knut och Alice Wallenbergs Stiftelse, KAW 2012.0078
    Tillgänglig från: 2016-11-17 Skapad: 2016-11-17 Senast uppdaterad: 2018-10-12Bibliografiskt granskad
    2. Factors Affecting Peptide Interactions with Surface-Bound Microgels
    Öppna denna publikation i ny flik eller fönster >>Factors Affecting Peptide Interactions with Surface-Bound Microgels
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    2016 (Engelska)Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 17, nr 2, s. 669-678Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Effects of electrostatics and peptide size on peptide interactions with surface-bound microgels were investigated with ellipsometry, confocal microscopy, and atomic force microscopy (AFM). Results show that binding of cationic poly-l-lysine (pLys) to anionic, covalently immobilized, poly(ethyl acrylate-co-methacrylic acid) microgels increased with increasing peptide net charge and microgel charge density. Furthermore, peptide release was facilitated by decreasing either microgel or peptide charge density. Analogously, increasing ionic strength facilitated peptide release for short peptides. As a result of peptide binding, the surface-bound microgels displayed pronounced deswelling and increased mechanical rigidity, the latter quantified by quantitative nanomechanical mapping. While short pLys was found to penetrate the entire microgel network and to result in almost complete charge neutralization, larger peptides were partially excluded from the microgel network, forming an outer peptide layer on the microgels. As a result of this difference, microgel flattening was more influenced by the lower Mw peptide than the higher. Peptide-induced deswelling was found to be lower for higher Mw pLys, the latter effect not observed for the corresponding microgels in the dispersed state. While the effects of electrostatics on peptide loading and release were similar to those observed for dispersed microgels, there were thus considerable effects of the underlying surface on peptide-induced microgel deswelling, which need to be considered in the design of surface-bound microgels as carriers of peptide loads, for example, in drug delivery or in functionalized biomaterials.

    Ort, förlag, år, upplaga, sidor
    American Chemical Society (ACS), 2016
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Farmaceutisk fysikalisk kemi
    Identifikatorer
    urn:nbn:se:uu:diva-278894 (URN)10.1021/acs.biomac.5b01616 (DOI)000369875900029 ()26750986 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet
    Tillgänglig från: 2016-02-26 Skapad: 2016-02-26 Senast uppdaterad: 2018-10-12Bibliografiskt granskad
    3. Membrane interactions of microgels as carriers of antimicrobial peptides
    Öppna denna publikation i ny flik eller fönster >>Membrane interactions of microgels as carriers of antimicrobial peptides
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    2018 (Engelska)Ingår i: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 513, s. 141-150Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Microgels are interesting as potential delivery systems for antimicrobial peptides. In order to elucidate membrane interactions of such systems, we here investigate effects of microgel charge density on antimicrobial peptide loading and release, as well as consequences of this for membrane interactions and antimicrobial effects, using ellipsometry, circular dichroism spectroscopy, nanoparticle tracking analysis, dynamic light scattering and z-potential measurements. Anionic poly(ethyl acrylate-co-methacrylic acid) microgels were found to incorporate considerable amounts of the cationic antimicrobial peptides LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW) and to protect incorporated peptides from degradation by infection-related proteases at high microgel charge density. As a result of their net negative z-potential also at high peptide loading, neither empty nor peptide-loaded microgels adsorb at supported bacteria-mimicking membranes. Instead, membrane disruption is mediated almost exclusively by peptide release. Mirroring this, antimicrobial effects against several clinically relevant bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa) were found to be promoted by factors facilitating peptide release, such as decreasing peptide length and decreasing microgel charge density. Microgels were further demonstrated to display low toxicity towards erythrocytes. Taken together, the results demonstrate some interesting opportunities for the use of microgels as delivery systems for antimicrobial peptides, but also highlight several key factors which need to be controlled for their successful use.

    Ort, förlag, år, upplaga, sidor
    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2018
    Nyckelord
    Antimicrobial peptide, Drug delivery, Lipid membrane, Microgel
    Nationell ämneskategori
    Fysikalisk kemi
    Identifikatorer
    urn:nbn:se:uu:diva-351759 (URN)10.1016/j.jcis.2017.11.014 (DOI)000428834900015 ()29145017 (PubMedID)
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, 604182
    Tillgänglig från: 2018-05-31 Skapad: 2018-05-31 Senast uppdaterad: 2018-10-12Bibliografiskt granskad
    4. Peptide-Loaded Microgels as Antimicrobial and Anti-Inflammatory Surface Coatings
    Öppna denna publikation i ny flik eller fönster >>Peptide-Loaded Microgels as Antimicrobial and Anti-Inflammatory Surface Coatings
    2018 (Engelska)Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, nr 8, s. 3456-3466Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Here we report on covalently immobilized poly(ethyl acrylate- co-methacrylic acid) microgels loaded with the host defense peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), which is derived from human heparin cofactor II, as well as its poly(ethylene glycol)-conjugated (PEGylated) version, KYE28PEG. Peptide loading and release, as well as the consequences of these processes on the microgel and peptide properties, were studied by in situ ellipsometry, confocal microscopy, zeta potential measurements, and circular dichroism spectroscopy. The results show that the microgel-peptide interactions are electrostatically dominated, thus promoted at higher microgel charge density, while PEGylation suppresses peptide binding. PEGylation also enhances the α-helix induction observed for KYE28 upon microgel incorporation. Additionally, peptide release is facilitated at physiological salt concentration, particularly so for KYE28PEG, which illustrates the importance of electrostatic interactions. In vitro studies on Escherichia coli show that the microgel-modified surfaces display potent antifouling properties in both the absence and presence of the incorporated peptide. While contact killing dominates at low ionic strength for the peptide-loaded microgels, released peptides also provide antimicrobial activity in bulk at a high ionic strength. Additionally, KYE28- and KYE28PEG-loaded microgels display anti-inflammatory effects on human monocytes. Taken together, these results not only show that surface-bound microgels offer an interesting approach for local drug delivery of host defense peptides but also illustrate the need to achieve high surface loads of peptides for efficient biological effects.

    Ort, förlag, år, upplaga, sidor
    American Chemical Society (ACS), 2018
    Nationell ämneskategori
    Farmakologi och toxikologi Biomaterialvetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-358186 (URN)10.1021/acs.biomac.8b00776 (DOI)000441852400029 ()29976055 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet, 2016-05157 2017-02341
    Tillgänglig från: 2018-08-24 Skapad: 2018-08-24 Senast uppdaterad: 2018-11-06Bibliografiskt granskad
    5. Microgels as carriers of antimicrobial peptides – effects of peptide PEGylation
    Öppna denna publikation i ny flik eller fönster >>Microgels as carriers of antimicrobial peptides – effects of peptide PEGylation
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-361401 (URN)
    Tillgänglig från: 2018-09-24 Skapad: 2018-09-24 Senast uppdaterad: 2018-10-12
    6. Avidin-biotin cross-linked microgel multilayers as carriers for antimicrobial peptides
    Öppna denna publikation i ny flik eller fönster >>Avidin-biotin cross-linked microgel multilayers as carriers for antimicrobial peptides
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    (Engelska)Ingår i: Artikel i tidskrift (Refereegranskat) Submitted
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-361399 (URN)
    Tillgänglig från: 2018-09-24 Skapad: 2018-09-24 Senast uppdaterad: 2018-10-12
  • 5.
    Dubbelboer, Ilse R
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Biopharmaceutical investigations of doxorubicin formulations used in liver cancer treatment: Studies in healthy pigs and liver cancer patients, combined with pharmacokinetic and biopharmaceutical modelling2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    There are currently two types of drug formulation in clinical use in the locoregional treatment of intermediate hepatocellular carcinoma (HCC). In the emulsion LIPDOX, the cytostatic agent doxorubicin (DOX) is dissolved in the aqueous phase, which is emulsified with the oily contrast agent Lipiodol® (LIP). In the microparticular system DEBDOX, DOX is loaded into the drug-eluting entity DC Bead™.

    The overall aim of the thesis was to improve pharmaceutical understanding of the LIPDOX and DEBDOX formulations, in order to facilitate the future development of novel drug delivery systems. In vivo release of DOX from the formulations and the disposition of DOX and its active metabolite doxorubicinol (DOXol) were assessed in an advanced multisampling-site acute healthy pig model and in patients with HCC. The release of DOX and disposition of DOX and DOXol where further analysed using physiologically based pharmacokinetic (PBPK) and biopharmaceutical (PBBP) modelling. The combination of in vivo investigations and in silico modelling could provide unique insight into the mechanisms behind drug release and disposition.

    The in vivo release of DOX from LIPDOX is not extended and controlled, as it is from DEBDOX. With both formulations, DOX is released as a burst during the early phase of administration. The in vivo release of DOX from LIPDOX was faster than from DEBDOX in both pigs and patients. The release from DEBDOX was slow and possibly incomplete. The in vivo release of DOX from LIPDOX and DEBDOX could be described by using the PBBP model in combination with in vitro release profiles.

    The disposition of DOX and DOXol was modelled using a semi-PBPK model containing intracellular binding sites. The contrast agent Lipiodol® did not affect the hepatobiliary disposition of DOX in the pig model. The control substance used in this study, cyclosporine A, inhibited the biliary excretion of DOX and DOXol but did not alter metabolism in healthy pigs. The disposition of DOX is similar in healthy pigs and humans, which was shown by the ease of translation of the semi-PBPK pig model to the human PBBP model.

    Delarbeten
    1. The Effects of Lipiodol and Cyclosporin A on the Hepatobiliary Disposition of Doxorubicin in Pigs
    Öppna denna publikation i ny flik eller fönster >>The Effects of Lipiodol and Cyclosporin A on the Hepatobiliary Disposition of Doxorubicin in Pigs
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    2014 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 11, nr 4, s. 1301-1313Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Doxorubicin (DOX) emulsified in Lipiodol (LIP) is used as local palliative treatment for unresectable intermediate stage hepatocellular carcinoma. The objective of this study was to examine the poorly understood effects of the main excipient in the drug delivery system, LIP, alone or together with cyclosporin A (CsA), on the in vivo liver disposition of DOX. The advanced, multi-sampling-site, acute pig model was used; samples were collected from three blood vessels (v. portae, v. hepatica and v. femoralis), bile and urine. The four treatment groups (TI-TIV) all received two intravenous 5 min infusions of DOX into an ear vein: at 0 and 200 min. Before the second dose, the pigs received a portal vein infusion of saline (TI), LIP (TII), CsA (TIII) or LIP and CsA (TIV). Concentrations of DOX and its active metabolite doxorubicinol (DOXol) were analyzed using UPLC-MS/MS. A multi-compartment model was developed to describe the distribution of DOX and DOXol in plasma, bile and urine. LIP did not affect the pharmacokinetics of DOX or DOXol. CsA (TIII and TIV) had no effect on the plasma pharmacokinetics of DOX, but a 2-fold increase in exposure to DOXol and a significant decrease in hepatobiliary clearance of DOX and DOXol was observed. Model simulations supported that CsA inhibits 99% of canalicular biliary secretion of both DOX and DOXol, but does not affect the metabolism of DOX to DOXol. In conclusion, LIP did not interact with transporters, enzymes and/or biological membranes important for the hepatobiliary disposition of DOX.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-222282 (URN)10.1021/mp4007612 (DOI)000334092700022 ()24558959 (PubMedID)
    Tillgänglig från: 2014-04-09 Skapad: 2014-04-09 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
    2. In vivo Drug Delivery Performance of Lipiodol-based Emulsion or Drug-eluting Beads in Patients with Hepatocellular Carcinoma
    Öppna denna publikation i ny flik eller fönster >>In vivo Drug Delivery Performance of Lipiodol-based Emulsion or Drug-eluting Beads in Patients with Hepatocellular Carcinoma
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    2017 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 14, nr 2, s. 448-458Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Doxorubicin (DOX) delivered in a lipiodol-based emulsion (LIPDOX) or in drug-eluting beads (DEBDOX) is used as palliative treatment in patients with intermediate-stage hepatocellular carcinoma (HCC). The primary objective of this study was to evaluate the in vivo delivery performance of DOX from LIPDOX or DEBDOX in HCC patients using the local and systemic pharmacokinetics of DOX and its main metabolite doxorubicinol (DOXol). Urinary excretion of DOX and DOXol, and their short-term safety and anti-tumor effects were also evaluated. In this open, prospective, non-randomized multi-center study, LIPDOX (n=13) or DEBDOX (n=12) were injected into the feeding arteries of the tumor. Local (vena cava/hepatic vein orifice) and systemic (peripheral vein) plasma concentrations of DOX and DOXol were determined in samples obtained up to 6 h and 7 days after treatment. Tumor response was assessed using computed tomography or magnetic resonance imaging. The Cmax and AUC0-24 h for DOX were 5.6-fold and 2.4-fold higher in LIPDOX vs DEBDOX recipients, respectively (p <0.001). After 6 h, the respective mean proportions of the dose remaining in the liver or drug-delivery system (DDS) were 49% for LIPDOX and 88% for DEBDOX. LIPDOX releases DOX faster than DEBDOX in HCC patients and provides more extensive local and systemic exposure (AUC) to DOX and DOXol initially (0-7 days). DEBDOX formulation has a release and distribution of DOX that is more restricted and rate controlled than LIPDOX.

    Nyckelord
    doxorubicin, doxorubicinol, drug eluting beads, local delivery, local therapy, hepatocellular carcinoma, liver cancer, lipiodol, transarterial chemoembolization, transarterial infusion chemotherapy
    Nationell ämneskategori
    Samhällsfarmaci och klinisk farmaci
    Identifikatorer
    urn:nbn:se:uu:diva-311314 (URN)10.1021/acs.molpharmaceut.6b00886 (DOI)000393630100012 ()27997198 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet, S21-2011-373
    Tillgänglig från: 2016-12-22 Skapad: 2016-12-22 Senast uppdaterad: 2018-01-13Bibliografiskt granskad
    3. A Model -Based Approach To Assessing the Importance of Intracellular Binding Sites in Doxorubicin Disposition
    Öppna denna publikation i ny flik eller fönster >>A Model -Based Approach To Assessing the Importance of Intracellular Binding Sites in Doxorubicin Disposition
    2017 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 14, nr 3, s. 686-698Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Doxorubicin is an anticancer agent, which binds reversibly to topoisomerase I and II, intercalates to DNA base pairs, and generates free radicals. Doxorubicin has a high tissue:plasma partition coefficient and high intracellular binding to the nucleus and other subcellular compartments. The metabolite doxorubicinol has an extensive tissue distribution. This porcine study investigated whether the traditional implementation of tissue binding, described by the tissue:plasma partition coefficient (K-p,K-t),could be used to appropriately analyze and/or simulate tissue doxorubicin and doxorubicinol concentrations in healthy pigs, when applying a physiologically based pharmacokinetic (PBPK) model approach, or whether intracellular binding is required in the semi-PBPK model. Two semi-PBPK models were developed and evaluated using doxorubicin and doxorubicinol concentrations in healthy pig blood, bile, and urine and kidney and liver tissues. In the generic semi-PBPK model, tissue binding was described using the conventional K-p,K-t approach. In the binding-specific semi-PBPK model, tissue binding was described using intracellular binding sites. The best semi-PBPK model was validated against a second data set of healthy pig blood and bile concentrations. Both models could be used for analysis and simulations of biliary and urinary excretion of doxorubicin and doxorubicinol and plasma doxorubicinol concentrations in pigs, but the binding-specific model was better at describing plasma doxorubicin concentrations. Porcine tissue concentrations were 400- to 1250-fold better captured by the binding-specific model. This model adequately predicted plasma doxorubicin concentration time and biliary doxorubicin excretion profiles against the validation data set. The semi-PBPK models applied were similarly effective for analysis of plasma concentrations and biliary and urinary excretion of doxorubicin and doxorubicinol in healthy pigs. Inclusion of intracellular binding in the doxorubicin semi-PBPK models was important to accurately describe tissue concentrations during in vivo conditions.

    Ort, förlag, år, upplaga, sidor
    AMER CHEMICAL SOC, 2017
    Nyckelord
    doxorubicin, physiologically based pharmacokinetic modeling, PBPK, pig
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-320394 (URN)10.1021/acs.molpharmaceut.6b00974 (DOI)000395847000012 ()28182434 (PubMedID)
    Tillgänglig från: 2017-04-20 Skapad: 2017-04-20 Senast uppdaterad: 2018-01-13Bibliografiskt granskad
    4. Porcine and human in vivo predictions for doxorubicin containing formulations used in locoregional HCC treatment
    Öppna denna publikation i ny flik eller fönster >>Porcine and human in vivo predictions for doxorubicin containing formulations used in locoregional HCC treatment
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-331567 (URN)
    Tillgänglig från: 2017-10-15 Skapad: 2017-10-15 Senast uppdaterad: 2018-01-13
  • 6.
    Pazesh, Samaneh
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Process-induced disorder of pharmaceutical materials: Mechanisms and quantification of disorder2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    One of the most important prerequisites in the drug development is to attain a reproducible and robust product in terms of its nature, and its chemical and physical properties. This can be challenging, since the crystalline form of drugs and excipients can be directly transformed into the amorphous one during normal pharmaceutical processing, referred to as process-induced amorphisation or process-induced disorder. The intention of this thesis was to address the mechanisms causing disorder during powder flow and milling and, in association with this, to evaluate, the ability of Raman spectroscopy and atomic force microscopy (AFM) to quantify and characterize process-induced disorder.

    The amorphisation mechanisms were controlled by stress energy distribution during processing, which in turn was regulated by a series of process parameters. Compression and shearing stress caused by sliding were stress types that acted on the particles during powder flow and ball milling process. However, sliding was the most important inter-particulate contact process giving rise to amorphisation and the transformation was proposed to be caused by vitrification. The plastic stiffness and elastic stiffness of the milling-induced particles were similar to a two-state particle model, however the moisture sorption characteristics of these particles were different. Thus the milled particles could not be described solely by a two-state particle model with amorphous and crystalline domains. 

    Raman spectroscopy proved to be an appropriate and effective technique in the quantification of the apparent amorphous content of milled lactose powder. The disordered content below 1% could be quantified with Raman spectroscopy. AFM was a useful approach to characterize disorder on the particle surfaces.

    In summary, this thesis has provided insight into the mechanisms involved in process-induced amorphisation of pharmaceutical powders and presented new approaches for quantification and characterization of disordered content by Raman spectroscopy and atomic force microscopy.

    Delarbeten
    1. Mechanism of Amorphisation of Micro-Particles of Griseofulvin During Powder Flow in a Mixer
    Öppna denna publikation i ny flik eller fönster >>Mechanism of Amorphisation of Micro-Particles of Griseofulvin During Powder Flow in a Mixer
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    2013 (Engelska)Ingår i: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 102, nr 11, s. 4036-4045Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The purpose of the research was to investigate the degree of solid-state amorphisation during powder flow and to propose a mechanism for this transformation. Micro-particles of griseofulvin (about 2m in diameter) were mixed in a shear mixer under different conditions to influence the inter-particulate collisions during flow, and the degree of amorphisation was determined by micro-calorimeter. The amorphisation of griseofulvin particles (GPs)during repeated compaction was also determined. The GPs generally became disordered during mixing in a range from about 6% to about 86%. The degree of amorphisation increased with increased mixing time and increased batch size of the mixer, whereas the addition of a lubricant to the blend reduced the degree of amorphisation. Repeated compaction using the press with ejection mode gave limited amorphisation, whereas repeated compaction without an ejection process gave minute amorphisation. It is concluded that during powder flow, the most important inter-particulate contact process that cause the transformation of a crystalline solid into an amorphous state is sliding. On the molecular scale, this amorphisation is proposed to be caused by vitrification, that is the melting of a solid because of the generation of heat during sliding followed by solidification into an amorphous phase.

    Nyckelord
    powder technology, powder flow, compaction, mixing, amorphisation, mechanical activation, vitrification, crystal defect, friction, sliding
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-210559 (URN)10.1002/jps.23713 (DOI)000325550400019 ()
    Tillgänglig från: 2013-11-13 Skapad: 2013-11-11 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
    2. Comminution-amorphisation relationships during ball milling of lactose at different milling conditions
    Öppna denna publikation i ny flik eller fönster >>Comminution-amorphisation relationships during ball milling of lactose at different milling conditions
    2017 (Engelska)Ingår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 528, nr 1-2, s. 215-227Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The purpose of the study was to investigate the relationship between comminution and amorphisation of alpha-lactose monohydrate particles during ball milling under different milling conditions, including ball-to-powder mass ratio, milling time and ball diameter. The results revealed that at a constant ball filling ratio, ball-to-powder mass ratio of 25:1 resulted in the lowest minimum particle diameter of similar to 5 mu m and the highest degree of apparent amorphous content of 82%. The rate of comminution was high during early stage of milling whereas the degree of apparent amorphous content increased gradually at a slow rate. An increased ball-to-powder mass ratio during milling increased both the rate of comminution and the rate of amorphisation. Using a given ball-to-powder mass ratio, the ball diameter affected the degree of apparent amorphous content of the particles while the particle diameter remained unchanged. The relationship between comminution and amorphisation could be described as consisting of two stages, i.e. comminution dominated and amorphisation dominated stage. It was proposed that the rate constant of comminution and amorphisation are controlled by stress energy distribution in the milling jar and the stress energy distribution is regulated by the ball motion pattern that can be affected by the process parameter used.

    Nyckelord
    Ball milling, process-induced disordering, amorphisation, comminution, comminution rate constant, α-lactose monohydrate, stress energy distribution
    Nationell ämneskategori
    Kemi
    Forskningsämne
    Farmaceutisk vetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-317797 (URN)10.1016/j.ijpharm.2017.05.043 (DOI)000408007600020 ()28546073 (PubMedID)
    Anmärkning

    Title in dissertation list of articles: "Comminution-amorphisation relationships during ball milling of lactose at different stress energies"

    Tillgänglig från: 2017-03-19 Skapad: 2017-03-19 Senast uppdaterad: 2017-11-27Bibliografiskt granskad
    3. Effect of milling on the plastic and the elastic stiffness of lactose particles
    Öppna denna publikation i ny flik eller fönster >>Effect of milling on the plastic and the elastic stiffness of lactose particles
    2018 (Engelska)Ingår i: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 114, s. 138-145Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The purpose of this study was to investigate the effect of degree of amorphisation of a series of lactose powders, prepared by milling α-lactose monohydrate powders for different time periods, on the plastic stiffness of the particles. As references, a series of physical mixtures consisting of original crystalline particles and amorphous particles obtained by spray-drying was used. In addition, the effect of powder pre-storage humidity on the mechanical properties was investigated. The particle plastic stiffness was assessed by the Heckel yield pressure derived from the relationship between porosity of the powder column and the applied compression pressure during confined powder compression.

     

    For milled particles of a low degree of disorder, a decreased particle size increased the particle plastic stiffness. For milled particles of constant particle size, the plastic stiffness decreased with an increased degree of disorder while the elastic stiffness seemed independent of the degree of disorder. The presence of moisture caused a recrystallization of milled particles with low degree of disorder which increased their plastic stiffness.

     

    For the physical mixtures of crystalline and amorphous particles, similar relationships between plastic stiffness and amorphous content as for the milled powders were obtained. A reasonable explanation is that the nature of the milled particles is represented by a two-state system with crystalline and amorphous domains.

    Nyckelord
    Milling-induced disorder, plasticity, elasticity, compression, amorphous lactose, Raman spectroscopy
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-317799 (URN)10.1016/j.ejps.2017.12.001 (DOI)000424977500014 ()29217203 (PubMedID)
    Anmärkning

    Title in dissertation list of references: Influence of degree of disorder on the Heckel yield pressure: a comparison between milled and physical mixtures of lactose

    Tillgänglig från: 2017-03-19 Skapad: 2017-03-19 Senast uppdaterad: 2018-09-10Bibliografiskt granskad
    4. Considerations on the quantitative analysis of apparent amorphicity of milled lactose by Raman spectroscopy
    Öppna denna publikation i ny flik eller fönster >>Considerations on the quantitative analysis of apparent amorphicity of milled lactose by Raman spectroscopy
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    2016 (Engelska)Ingår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 511, nr 1, s. 488-504Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The main purpose of the study was to evaluate various pre-processing and quantification approaches of Raman spectrum to quantify low level of amorphous content in milled lactose powder. To improve the quantification analysis, several spectral pre-processing methods were used to adjust background effects. The effects of spectral noise on the variation of determined amorphous content were also investigated theoretically by propagation of error analysis and were compared to the experimentally obtained values. Additionally, the applicability of calibration method with crystalline or amorphous domains in the estimation of amorphous content in milled lactose powder was discussed. Two straight baseline pre-processing methods gave the best and almost equal performance. By the succeeding quantification methods, PCA performed best, although the classical least square analysis (CLS) gave comparable results, while peak parameter analysis displayed to be inferior. The standard deviations of experimental determined percentage amorphous content were 0.94% and 0.25% for pure crystalline and pure amorphous samples respectively, which was very close to the standard deviation values from propagated spectral noise. The reasonable conformity between the milled samples spectra and synthesized spectra indicated representativeness of physical mixtures with crystalline or amorphous domains in the estimation of apparent amorphous content in milled lactose.

    Nyckelord
    Raman spectroscopy, Lactose, Amorphous content, Spectral data analysis, Principal component analysis (PCA), Milling induced disorder
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-304150 (URN)10.1016/j.ijpharm.2016.07.001 (DOI)000382263700051 ()27397869 (PubMedID)
    Tillgänglig från: 2016-10-03 Skapad: 2016-10-03 Senast uppdaterad: 2018-01-14Bibliografiskt granskad
    5. Determination of Interfacial Amorphicity in Functional Powders
    Öppna denna publikation i ny flik eller fönster >>Determination of Interfacial Amorphicity in Functional Powders
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    2017 (Engelska)Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 33, nr 4, s. 920-926Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The nature of the surfaces of particles of pharmaceutical ingredients, food powders, and polymers is a determining factor for their performance in for example tableting, powder handling, or mixing. Changes on the surface structure of the material will impact the flow properties, dissolution rate, and tabletability of the 2 powder blend. For crystalline materials, surface amorphization is a phenomenon which is known to impact performance. Since it is important to measure and control the level of amorphicity, several characterization techniques are available to determine the bulk amorphous content of a processed material. The possibility of characterizing the degree of amorphicity at the surface, for example by studying the mechanical properties of the particles' surface at the nanoscale, is currently only offered by atomic force microscopy (AFM). The AFM PeakForce QNM technique has been used to measure the variation in energy dissipation (eV) at the surface of the particles which sheds light on the mechanical changes occurring as a result of amorphization or recrystallization events. Two novel approaches for the characterization of amorphicity are presented here. First, since particles are heterogeneous, we present a methodology to present the results of extensive QNM analysis of multiple particles in a coherent and easily interpreted manner, by studying cumulative distributions of dissipation data with respect to a threshold value which can be used to distinguish the crystalline and amorphous states. To exemplify the approach, which is generally applicable to any material, reference materials of purely crystalline alpha-lactose monohydrate and completely amorphous spray dried lactose particles were compared to a partially amorphized alpha-lactose monohydrate sample. Dissipation data are compared to evaluations of the lactose samples with conventional AFM and SEM showing significant topographical differences. Finally, the recrystallization of the surface amorphous regions in response to humidity was followed by studying the dissipation response of a well-defined surface region over time, which confirms both that dissipation measurement is a useful measure of surface amorphicity and that significant recrystallization occurs at the surface in response to humidity.

    Nationell ämneskategori
    Kemi
    Identifikatorer
    urn:nbn:se:uu:diva-317609 (URN)10.1021/acs.langmuir.6b03969 (DOI)000393269700010 ()
    Tillgänglig från: 2017-03-16 Skapad: 2017-03-16 Senast uppdaterad: 2017-11-29Bibliografiskt granskad
  • 7.
    Thunander Sundbom, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    The influence of gender and depression on drug utilization: Pharmacoepidemiological research in Sweden2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Background Drug use has increased over recent decades, and is especially great among women and among people with mental health problems. To take advantage of the full potential of drugs and to avoid drug-related problems, drug prescription needs to be correct and the drugs need to be taken according to the prescribed regimens. Research on drug utilization is thus important to the public health.

    Aim To study the influence of gender and depression on drug utilization, prescription of drugs and self-reported use of drugs, i.e. adherence.

    Methods The thesis included two population-based questionnaires and data from the Swedish Prescribed Drug Register (SPDR) covering Swedish citizens 18-84 years. The questionnaire in Study I and II included items on prescription drug use and adherence to treatment regimens; Study II also included the Hospital Anxiety and Depression Scale (HADS) for self-estimation of anxiety/depression. The questionnaire in Study III included the HADS and data from the SPDR on prescribed antidepressants. Study IV included data from the SPDR on all types of prescribed drugs.

    Results Men and women differed in non-adherent behaviours and reasons for non-adherence, for example, men were more likely to report forgetting to take the drug, while women were more likely to report adverse drug reactions (ADRs) as a reason for non-adherence. Further, both anxiety and depression were associated with non-adherence and with ADRs as a reason for non-adherence. In addition, men reported depression to a greater extent than women did but used antidepressants to a lesser extent, while women used antidepressants without reporting depression more often than men did, which may be a sign of under-treatment among men and over-treatment among women. Moreover, the associations between antidepressants and other types of drugs differed by gender; they were often specific, or stronger, in women than in men, which may be a sign of a gender difference in comorbidity between depression and other conditions.

    Conclusions Although the cross-sectional study design prevented confirmation of causality, the thesis found that gender and depression influence both prescription of drugs and adherence, and are thus important to pay attention to in clinical practice as well as research.

    Delarbeten
    1. Women and men report different behaviours in, and reasons for medication non-adherence: a nationwide Swedish survey
    Öppna denna publikation i ny flik eller fönster >>Women and men report different behaviours in, and reasons for medication non-adherence: a nationwide Swedish survey
    2012 (Engelska)Ingår i: Pharmacy Practice, ISSN 1885-642X, E-ISSN 1886-3655, Vol. 10, nr 4, s. 207-221Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Objectives

    The aim of the present study was to analyse gender differences in self-reported non-adherence (NA) to prescribed medication in the Swedish general population. We aimed to study unintentional and intentional NA as well as the reasons given for NA.

    Methods

    A questionnaire was mailed to a cross-sectional, random, national sample of people aged 18-84 years in Sweden (n=7985). The response rate was 61.1% (n=4875). The questionnaire covered use of prescription drugs, NA behaviourand reasons for NA.

    Results

    Use of prescription drugs was reported by 59.5% (n=2802) of the participants, and 66.4% (n=1860) of these participants did not adhere to the prescribed regimen. No overall gender differences in reporting NA were found. However, when analysing the various types of NA behaviour and the reasons for NA, different gender patterns emerged. Men were more likely to report forgetting [OR 0.77 (95% CI 0.65:0.92)], changing the dosage [OR 0.64 (95% CI 0.52:0.79)] and that they had recovered [14.3%, (OR 0.71 (95% CI 0.56:0.90)] as a reason. In contrast, more women than men reported filling the prescription but not taking the drug [OR 1.25 (95% CI 1.02:1.54)] and reported the development of adverse drug reactions (ADRs) [OR 1.89 (95% CI 1.37:2.59)] as a reason more commonly. The gender differences remained, in most cases, after controlling for confounders such as age, socioeconomic factors, medical problems and attitudes toward drugs.

    Conclusions

    Women and men have different patterns of NA behaviour and different reasons for NA. Therefore, if adherence is to be improved, a wide knowledge of all the reasons for NA is required, along with an understanding of the impact of gender on the outcomes.

     

    Nyckelord
    Medication Adherence, Health knowledge, Attitudes, Practice, Health care surveys, Sweden
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Farmakoepidemiologi
    Identifikatorer
    urn:nbn:se:uu:diva-193611 (URN)
    Tillgänglig från: 2013-02-05 Skapad: 2013-02-05 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
    2. The influence of symptoms of anxiety and depression on medication nonadherence and its causes: a population based survey of prescription drug users in Sweden
    Öppna denna publikation i ny flik eller fönster >>The influence of symptoms of anxiety and depression on medication nonadherence and its causes: a population based survey of prescription drug users in Sweden
    2013 (Engelska)Ingår i: Patient Preference and Adherence, ISSN 1177-889X, E-ISSN 1177-889X, Vol. 7, s. 805-811Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Purpose: The purpose of this study was to evaluate the associations between self-reported symptoms of anxiety and/or depression, nonadherent (NA) behaviors, and reasons for NA to medication regimens.

    Methods: A population based cross-sectional study with questionnaire was performed in the general Swedish population. The participants were 2802 prescription drug users aged 18-84 years. The questionnaire covered use of prescription drugs, symptoms of anxiety and/or depression, based on the Hospital Anxiety and Depression Scale (HADS), various NA behavior types, intentional and unintentional, and various reasons for NA.

    Results: Symptoms of anxiety and depression, independently and in combination, were associated with unintentional and intentional NA, with a stronger association with intentional NA. Regarding the reasons given for NA, for example anxiety, independently or in combination with depression, was associated with a fear of developing adverse drug reactions (ADRs). Depression, independently or in combination with anxiety, on the other hand, was associated with the actual development of ADRs.

    Conclusion: A cross-sectional design such as this does not allow assessment of causality derived from the results. However, the results indicate that patients experiencing symptoms of psychological distress are at increased risk of NA, especially intentional NA, and could therefore benefit from extra attention from the health care professional. Patients with symptoms of anxiety and/or depression should be identified and monitored for the development and/or fear of ADRs, in order to improve adherence to medication regimens.

    Nyckelord
    Medication Adherence, Anxiety symptoms, Depression symptoms, Reasons
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Forskningsämne
    Farmakoepidemiologi
    Identifikatorer
    urn:nbn:se:uu:diva-205574 (URN)10.2147/PPA.S50055 (DOI)000323147600001 ()
    Tillgänglig från: 2013-08-20 Skapad: 2013-08-20 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
    3. Are men under-treated and women over-treated with antidepressants?: Findings from a cross-sectional survey in Sweden
    Öppna denna publikation i ny flik eller fönster >>Are men under-treated and women over-treated with antidepressants?: Findings from a cross-sectional survey in Sweden
    2017 (Engelska)Ingår i: BJPsych bulletin, ISSN 2056-4694, E-ISSN 2056-4708, Vol. 41, nr 3, s. 145-150Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Aims and method: To examine gender differences in self-reported depression and prescribed antidepressants (ADs). The Hospital Anxiety and Depression Scale was used to assess depression, and information on prescribed ADs was obtained from the Swedish Prescribed Drug Register.

    Results: Depression was reported by 11.7% of the participants (12.3% men and 11.2% women). ADs were prescribed for 7.6% of the participants (5.3% men, 9.8% women). Among men, 1.8% reported depression and used ADs, 10.5% reported depression but did not use ADs, and 3.6% used ADs but did not report depression. The corresponding figures for women were 2.6%, 8.6% and 7.2%.

    Clinical implications: Men report depression to a greater extent than women but are prescribed ADs to a lesser extent, possibly a sign of under-treatment. Women are prescribed ADs without reporting depression more often than men, possibly a sign of over-treatment. Although the causes remain unclear, diagnostic and treatment guidelines should benefit from considering gender differences in these respects.

    Nyckelord
    Self-reported depression, Antidepressants, Gender differences, Over-treatment, Under-treatment
    Nationell ämneskategori
    Farmaceutiska vetenskaper Psykiatri
    Forskningsämne
    Farmakoepidemiologi
    Identifikatorer
    urn:nbn:se:uu:diva-310328 (URN)10.1192/pb.bp.116.054270 (DOI)000407961600004 ()28584650 (PubMedID)
    Tillgänglig från: 2016-12-14 Skapad: 2016-12-14 Senast uppdaterad: 2018-05-04Bibliografiskt granskad
    4. Gender differences in the association between prescribed antidepressants and other prescribed drugs: a nationwide register-based study in Sweden
    Öppna denna publikation i ny flik eller fönster >>Gender differences in the association between prescribed antidepressants and other prescribed drugs: a nationwide register-based study in Sweden
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Background: People with depression are prescribed more drugs than people in general, partly due to comorbidity with other conditions. However, little research has been done on depression-related drug use from a gender perspective.

    Aim: Examine gender differences in the association between antidepressants, other drugs, and polypharmacy.

    Methods: Data on drugs dispensed October to December 2016 to all Swedish citizens aged 18-84 years were collected from the Swedish prescribed drug register. Logistic regression analyses were performed to examine gender differences in the associations between antidepressants and other drugs.

    Results: For both men and women, associations were found between antidepressants and drugs for alimentary tract problems, respiratory problems, drugs for the blood, and drugs for the nervous system, analgesics, and polypharmacy. For many of the drugs, for example those for respiratory problems and analgesics, the association was stronger in women than in men. However, concerning drugs for the nervous system and polypharmacy, the association was stronger in men than in women. Furthermore, for women, but not men, associations were found for drugs for diabetes, musculoskeletal problems, dermatological problems, and systemic hormones.

    Conclusions: Many of the associations between antidepressants and other drugs were found to be specific, or stronger, among women than among men. In some cases, however, the associations were stronger in men. Whether this indicates that men and women differ in comorbidity between depression and other conditions cannot be concluded based on this cross-sectional study. However, physicians should be aware that possible gender differences in comorbidity exist, and because comorbidity between depression and other conditions impairs the possibility of recovery, and decreases adherence, screening for depression could be valuable.

    Nyckelord
    Gender, Antidepressants, Prescribed drugs, Polypharmacy
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Forskningsämne
    Farmakoepidemiologi
    Identifikatorer
    urn:nbn:se:uu:diva-328064 (URN)
    Tillgänglig från: 2017-08-16 Skapad: 2017-08-16 Senast uppdaterad: 2017-10-04Bibliografiskt granskad
  • 8.
    Singh, Shalini
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Amphiphilic Peptide Interactions with Complex Biological Membranes: Effect of peptide properties on antimicrobial and anti-inflammatory effects2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    With increasing problem of resistance development in bacteria against conventional antibiotics, as well as problems associated with diseases either triggered or enhanced by infection, there is an urgent need to identify new types of effective therapeutics for the treatment of infectious diseases and its consequences. Antimicrobial and anti-inflammatory peptides have attracted considerable interest as potential new antibiotics in this context. While antimicrobial function of such peptides is being increasingly understood demonstrated to be due to bacterial membrane disruption, the mechanisms of their anti-inflammatory function are poorly understood. Since bacterial membrane component lipopolysaccharide triggers inflammation, this thesis aims at clarifying importance of lipopolysaccharide (LPS)-peptide interactions while investigating possible modes of action of peptides exhibiting anti-inflammatory effect. Furthermore, effect of poly(ethylene)glycol (PEG)-conjugation was investigated to increase performance of such peptides.

    Results presented in this thesis demonstrate that peptide-induced LPS- and lipid A binding/scavenging is necessary but not sufficient criterium for anti-inflammatory effects of peptides. Furthermore, preferential binding to LPS over lipid membrane, as well as higher binding affinity to the lipid A moiety within LPS, are seen for these peptides. In addition, results demonstrate that apart from direct LPS scavenging, membrane-localized peptide-induced LPS scavenging seem to contribute partially to anti-inflammatory effect. Furthermore, fragmentation and densification of LPS aggregates, in turn dependent on the peptide secondary structure on LPS binding, as well as aromatic packing interactions, correlate to the anti-inflammatory effect, thus promoting peptide-induced packing transition in LPS aggregates as key for anti-inflammatory functionality. Thus, peptide-induced LPS aggregate disruption together with reduction of the negative charge of LPS suggests the importance of phagocytosis as an alternative to the inflammatory pathway, which needs to be further investigated. Furthermore, PEG conjugation of peptide results in strongly reduced toxicity at a cost of reduced antimicrobial activity but markedly retained anti-inflammatory effect.

    Taken together, the results obtained in this work have demonstrated several key issues which need to be taken into consideration in the development of effective and selective anti-inflammatory peptide therapeutics for the treatment of severe Gram-negative bacterial infections.

    Delarbeten
    1. Membrane and lipopolysaccharide interactions of C-terminal peptides from S1 peptidases
    Öppna denna publikation i ny flik eller fönster >>Membrane and lipopolysaccharide interactions of C-terminal peptides from S1 peptidases
    2012 (Engelska)Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, nr 9, s. 2244-2251Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The mechanisms underlying antimicrobial and anti-endotoxic effects were investigated for a series of structurally related peptides derived from the C-terminal region of S1 peptidases. For this purpose, results on bacterial killing were compared to those on peptide-induced liposome leakage, and to ellipsometry and dual polarization interferometry results on peptide binding to, and disordering of, supported lipid bilayers. Furthermore, the ability of these peptides to block endotoxic effects caused by bacterial lipopolysaccharide (LPS), monitored through NO production in macrophages, was compared to the binding of these peptides to LPS, and to secondary structure formation in the peptide/LPS complex. Bacteria killing, occurring through peptide-induced membrane lysis, was found to correlate with liposome rupture, and with the extent of peptide binding to the lipid membrane, no adsorption threshold for peptide insertion being observed. Membrane and LPS binding was found to depend on peptide net charge, illustrated by LPS binding increasing with increasing peptide charge, and peptides with net negative charge being unable to lyse membranes, kill bacteria, and block LPS-induced endotoxic effect. These effects were, however, also influenced by peptide hydrophobicity. LPS binding was furthermore demonstrated to be necessary, but not sufficient, for anti-endotoxic effect of these peptides. Circular dichroism spectroscopy showed that pronounced helix formation occurs in peptide/LPS complexes for all peptides displaying anti-endotoxic effect, hence potentially linked to this functionality. Similarly, ordered secondary structure formation was correlated to membrane binding, lysis, and antimicrobial activity of these peptides. Finally, preferential binding of these peptides to LPS over the lipid membrane was demonstrated.

    Nyckelord
    Antimicrobial peptide, Dual polarization interferometry, Ellipsometry, Lipopolysaccharide, Liposome, Membrane
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-180268 (URN)10.1016/j.bbamem.2012.03.017 (DOI)000306882600019 ()
    Tillgänglig från: 2012-09-03 Skapad: 2012-09-03 Senast uppdaterad: 2017-12-07Bibliografiskt granskad
    2. Lipopolysaccharide Interactions of C-Terminal Peptides from Human Thrombin
    Öppna denna publikation i ny flik eller fönster >>Lipopolysaccharide Interactions of C-Terminal Peptides from Human Thrombin
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    2013 (Engelska)Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, nr 5, s. 1482-1492Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRL-KKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-202380 (URN)10.1021/bm400150c (DOI)000319034600027 ()
    Tillgänglig från: 2013-06-24 Skapad: 2013-06-24 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
    3. Effects of linear amphiphilicity on membrane interactions of C-terminal thrombin peptides
    Öppna denna publikation i ny flik eller fönster >>Effects of linear amphiphilicity on membrane interactions of C-terminal thrombin peptides
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    2014 (Engelska)Ingår i: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 4, nr 71, s. 37582-37591Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Effects of linear amphiphilicity on membrane interactions of antimicrobial peptides were investigated by ellipsometry, dual polarization interferometry, fluorescence spectroscopy, light scattering, and circular dichroism. In doing so, the thrombin-derived GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) was compared to WFF25 (WFFFYYLIIGGGVVTHQQRKKKKDE) of identical composition, but with amino acids sorted according to hydrophobicity, the latter peptide thus displaying pronounced linear amphiphilicity. In addition, GKY25d (GKYG(f) YTH(v) FRL(k) KWI(q) KVI(d) QFGE; with an identical sequence but with selected D-amino acid substitutions) was included as a control peptide, for which conformationally induced (helix-related) amphiphilicity was suppressed. Through its pronounced linear amphiphilicity, WFF25, but not the less amphiphilic GKY25 and GKY25d, forms aggregates in solution. Through its terminal W/F stretch, WFF25 also displays pronounced selectivity, with higher membrane binding and liposome rupture than GKY25 and GKY25d for anionic membranes, but suppressed peptide insertion and lytic effects for zwitterionic ones. In addition, WFF25 binds extensively to anionic polyelectrolyte components in bacterial membranes, i.e., lipopolysaccharide and lipoteichoic acid, resulting in reduced antimicrobial effects through peptide scavenging, not seen for the less amphiphilic GKY25 and GKY25d peptides. Taken together, the results thus demonstrate a series of striking effects for highly amphiphilic peptides, which need to be recognized in the development of such compounds as potential peptide therapeutics.

    Nationell ämneskategori
    Klinisk medicin Kemi
    Identifikatorer
    urn:nbn:se:uu:diva-233621 (URN)10.1039/c4ra05420b (DOI)000341454600018 ()
    Tillgänglig från: 2014-10-07 Skapad: 2014-10-07 Senast uppdaterad: 2017-12-05Bibliografiskt granskad
    4. Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides
    Öppna denna publikation i ny flik eller fönster >>Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides
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    2013 (Engelska)Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, nr 11, s. 2709-2719Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLERKLTHRLFRRNEGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics. 

    Nyckelord
    Antimicrobial peptide, Dual polarization interferometry, Ellipsometry, Lipopolysaccharide, Liposome, Membrane
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-211434 (URN)10.1016/j.bbamem.2013.06.015 (DOI)000326143200038 ()
    Tillgänglig från: 2013-11-27 Skapad: 2013-11-25 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
    5. Effects of PEGylation on Membrane and Lipopolysaccharide Interactions of Host Defense Peptides
    Öppna denna publikation i ny flik eller fönster >>Effects of PEGylation on Membrane and Lipopolysaccharide Interactions of Host Defense Peptides
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    2014 (Engelska)Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, nr 4, s. 1337-1345Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Effects of poly(ethylene glycol) (PEG) conjugation on peptide interactions with lipid membranes and lipopolysaccharide (LPS) were investigated for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), an antimicrobial and anti-inflammatory peptide derived from human heparin cofactor II. In particular, effects of PEG length and localization was investigated by ellipsometry, circular dichroism, nanoparticle tracking analysis, and fluorescence/electron microscopy. PEGylation of KYE28 reduces peptide binding to lipid membranes, an effect accentuated at increasing PEG length, but less sensitive to conjugation site. The reduced binding causes suppressed liposome leakage induction, as well as bacterial lysis. As a result of this, the antimicrobial effects of KYE28 is partially lost with increasing PEG length, but hemolysis also strongly suppressed and selecticity improved. Through this, conditions can be found, at which the PEGylated peptide displays simultaneously efficient antimicrobial affects and low hemolysis in blood. Importantly, PEGylation does not markedly affect the anti-inflammatory effects of KYE28. The combination of reduced toxicity, increased selectivity, and retained anti-inflammatory effect after PEGylation, as well as reduced scavenging by serum proteins, thus shows that PEG conjugation may offer opportunities in the development of effective and selective anti-inflammatory peptides.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-225069 (URN)10.1021/bm401884e (DOI)000334571600026 ()
    Tillgänglig från: 2014-06-23 Skapad: 2014-05-27 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
    6. Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for use in Plant Disease Control
    Öppna denna publikation i ny flik eller fönster >>Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for use in Plant Disease Control
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    2016 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, nr 25, s. 13301-13317Artikel i tidskrift, Meeting abstract (Refereegranskat) Published
    Abstract [en]

    KYE28(KYEITTIHNLFRKLTHRLFRRNFGYTLR), the representative sequence  of helix D of heparin co-factor II, was demonstrated to be potent against agronomically important Gram-negative plant pathogens X. vesicatoria and X. oryzae,capable of inhibiting disease symptoms in detached tomato leaves. NMR studies in presence of lipopolysaccharide provided structural insights into the mechanisms underlying this, notably in relation to outer membrane permeabilisation. The three-dimensional solution structure of KYE28 in LPS is characterised by a N-ter helical segment, an intermediate loop and an extended C-ter. The two termini are in close proximity to each other via aromatic packing interactions, while the positively charged residues formed an exterior polar shell. To further demonstrate the importance of the aromatic residues for this, a mutant peptide KYE28A, with Ala substitutions at F11, F19, F23 and Y25 showed attenuated antimicrobial activity at high salt concentrations, as well as lower membrane disruption and LPS binding abilities compared to KYE28. In contrast to KYE28, KYE28A adopted an opened out helical structure in LPS with extended N- and C-ter and a small break in between the helical segments. Aromatic packing interactions were completely lost, although hydrophobic interaction between the side chains of hydrophobic residues were still partly retained, imparting an amphipathic character and explaining its residual antimicrobial activity and LPS binding as observed from ellipsometry and ITC. We thus present important structural aspects of KYE28, constituting an aromatic zipper, of potential importance, for the development of novel plant protection agents and therapeutic agents.

    Nyckelord
    LPS, Antimicrobial
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Forskningsämne
    Farmaceutisk fysikalisk kemi
    Identifikatorer
    urn:nbn:se:uu:diva-282778 (URN)10.1074/jbc.M116.719575 (DOI)000379770500033 ()27137928 (PubMedID)
    Tillgänglig från: 2016-04-06 Skapad: 2016-04-06 Senast uppdaterad: 2018-05-15Bibliografiskt granskad
  • 9.
    Mateus, André
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci. Faculty of Pharmacy, University of Lisbon.
    Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs.

    This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior.

    The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments.

    The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity.

    Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be active in cellular assays.

    In conclusion, the methodology can be used to predict if new drug candidates reach their intracellular targets in sufficient amounts. Furthermore, the methodology can improve in vitro predictions of drug clearance and drug-drug interactions, by measuring the drug available for intracellular enzymes. Finally, this work can be expanded to other xenobiotics, e.g., to predict their intracellular toxicity.

    Delarbeten
    1. Rapid Measurement of Intracellular Unbound Drug Concentrations
    Öppna denna publikation i ny flik eller fönster >>Rapid Measurement of Intracellular Unbound Drug Concentrations
    2013 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 10, nr 6, s. 2467-2478Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Intracellular unbound drug concentrations determine affinity to targets in the cell interior. However, due to difficulties in measuring them, they are often overlooked in pharmacology. Here we present a simple experimental technique for the determination of unbound intracellular drug concentrations in cultured cells that is based on parallel measurements of cellular drug binding and steady-state intracellular drug concentrations. Binding in HEK293 cells was highly correlated with binding in liver-derived systems, whereas binding in plasma did not compare well with cellular binding. Compound lipophilicity increased drug binding, while negative charge and aromatic functional groups decreased binding. Intracellular accumulation of unbound drug was consistent with pH dependent subcellular sequestration, as confirmed by modeling and by inhibition of subcellular pH gradients. The approach developed here can be used to measure intracellular unbound drug concentrations in more complex systems, for example, cell lines with controlled expression of transporters and enzymes or primary cells.

    Nyckelord
    intracellular unbound concentrations, drug binding, drug transport, drug accumulation, membrane partitioning
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-204292 (URN)10.1021/mp4000822 (DOI)000320015600037 ()
    Tillgänglig från: 2013-07-29 Skapad: 2013-07-29 Senast uppdaterad: 2018-07-30
    2. A High-Throughput Cell-Based Method to Predict the Unbound Drug Fraction in the Brain
    Öppna denna publikation i ny flik eller fönster >>A High-Throughput Cell-Based Method to Predict the Unbound Drug Fraction in the Brain
    2014 (Engelska)Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 57, nr 7, s. 3005-3010Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Optimization of drug efficacy in the brain requires understanding of the local exposure to unbound drug at the site of action. This relies on measurements of the unbound drug fraction (f(u,brain)), which currently requires access to brain tissue. Here, we present a novel methodology using homogenates of cultured cells for rapid estimation of f(u,brain). In our setup, drug binding to human embryonic kidney cell (HEK293) homogenate was measured in a small-scale dialysis apparatus. To increase throughput, we combined drugs into cassettes for simultaneous measurement of multiple compounds. Our method estimated f(u,brain) with an average error of 1.9-fold. We propose that our simple method can be used as an inexpensive, easily available and high-throughput alternative to brain tissues excised from laboratory animals. Thereby, estimates of unbound drug exposure can now implemented at a much earlier stage of the drug discovery process, when molecular property changes are easier to make.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-224730 (URN)10.1021/jm401963n (DOI)000334572000017 ()
    Tillgänglig från: 2014-05-22 Skapad: 2014-05-19 Senast uppdaterad: 2018-07-30
    3. Impact of drug transporters on intracellular drug concentrations
    Öppna denna publikation i ny flik eller fönster >>Impact of drug transporters on intracellular drug concentrations
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-276093 (URN)
    Tillgänglig från: 2016-02-09 Skapad: 2016-02-09 Senast uppdaterad: 2018-01-10
    4. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
    Öppna denna publikation i ny flik eller fönster >>CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
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    2016 (Engelska)Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, artikel-id 11040Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

    Nationell ämneskategori
    Farmakologi och toxikologi
    Identifikatorer
    urn:nbn:se:uu:diva-276077 (URN)10.1038/ncomms11040 (DOI)000372887500001 ()27010513 (PubMedID)
    Forskningsfinansiär
    Karolinska Institutets ForskningsstiftelseVetenskapsrådetCancerfondenKnut och Alice Wallenbergs Stiftelse
    Anmärkning

    Artursson, P., Martinez-Molina, D och Nordlund, P. delar sistaförfattarskapet.

    Tillgänglig från: 2016-02-09 Skapad: 2016-02-09 Senast uppdaterad: 2018-01-10Bibliografiskt granskad
    5. Impact of intracellular drug bioavailability on cellular drug response
    Öppna denna publikation i ny flik eller fönster >>Impact of intracellular drug bioavailability on cellular drug response
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Farmakologi och toxikologi
    Identifikatorer
    urn:nbn:se:uu:diva-276089 (URN)
    Tillgänglig från: 2016-02-09 Skapad: 2016-02-09 Senast uppdaterad: 2018-01-10
  • 10.
    Hellrup, Joel
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Pharmaceutical Nanocomposites: Structure–Mobility–Functionality Relationships in the Amorphous State2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Amorphous materials are found in pharmaceutical formulations both as excipients and active ingredients. Indeed, these formulations are becoming an essential strategy for incorporating drugs into well-performing solid dosage forms. However, there is an unmet need of better understanding of the microstructure and component interactions in amorphous formulations to be able to design materials with improved functionalities. The aim of this thesis is to give deepened knowledge about structure-mobility-functionality relationships in amorphous for-mulations by studying composites produced from sugars and filler particles. The structure, the mobility, and physical stability of the composite materials were studied using calorimetry, X-ray diffraction, microscopy, spectroscopy, and molecular dynamics simulations. Further, the moisture sorption of the composites was determined with dynamic vapor sorption. The compression mechanics of the composites was evaluated with compression analysis.

    It was demonstrated that fillers change the overall properties of the amorphous material. Specifically, the physical stability of the composite was by far improved compared to the amorphous sugar alone. This effect was pronounced for formulations with 60 wt% filler content or more. Amorphous lactose that normally recrystallizes within a few minutes upon humidity exposure, could withstand recrystallization for several months at 60% RH in composites with 80 wt% cellulose nanocrystals (CNC) or sodium montmorillonite (Na-MMT). The increased physical stability of the amorphous sugars was related to intra-particle confinement in extra-particle voids formed by the fillers and to immobilization of the amorphous phase at the surface of the fillers. Also, the composite formation led to increased particle hardness for the lactose/CNC and the lactose/Na-MMT nanocomposites. The largest effect on particle hardness was seen with 40-60 wt% nanofiller and could be related to skeleton formation of the nanofillers within the composite particles. The hygroscopicity for the lactose/Na-MMT nanocomposites decreased as much as 47% compared to ideal simple mixtures of the neat components. The nanofillers did not influence the water sorption capacity in the amorphous domains; however, lactose (intercalated into Na-MMT) interacted with the sodium ions in the interlayer space which led to the lowered hygroscopicity of this phase.

    The thesis advanced the knowledge of the microstructure of amorphous pharmaceutical com-posites and its relationship with pharmaceutical functionalities. It also presented new approaches for stabilizing the amorphous state by using fillers. The concept illustrated here might be used to understand similar phenomena of stabilization of amorphous formulations.

    Delarbeten
    1. Pharmaceutical micro-particles give amorphous sucrose higher physical stability
    Öppna denna publikation i ny flik eller fönster >>Pharmaceutical micro-particles give amorphous sucrose higher physical stability
    2011 (Engelska)Ingår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 409, nr 1-2, s. 96-103Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The aim of this study was to explore how pharmaceutical micro-sized filler particles affect the amorphous stability of sucrose in sucrose/filler particle composites produced by freeze-drying. Focus was put on the filler particles' properties crystallinity, hygroscopicity, hydrophobicity, and surface area, and their influence on physical stability of the amorphous phase. The micro-sized filler particles were examined with Blaine permeametry, gas adsorption, pycnometry, gravimetric vapour sorption, X-ray diffraction, and light microscopy before composites of sucrose and micro-sized filler particles were prepared by freeze-drying. The stability of the composites was examined with X-ray diffraction, differential scanning calorimetry (DSC), and microcalorimetry. All composites were amorphous and showed higher stability compared to pure amorphous sucrose, which was evident from a delay in heat and moisture-induced crystallization. However, calcium carbonate and oxazepam micro-sized filler particles lost their ability to stabilize the amorphous sucrose when exposed to humidity. The dry glass transition temperature (T-g) was higher for the composites, indicating the stabilization was mediated by a reduced molecular mobility of the amorphous phase.

    Nyckelord
    Amorphous, Sucrose, Physical stability, Freeze-drying, Crystallization, Micro-particles
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-153962 (URN)10.1016/j.ijpharm.2011.02.031 (DOI)000290135800012 ()21356288 (PubMedID)
    Tillgänglig från: 2011-05-23 Skapad: 2011-05-23 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
    2. Inhibition of Recrystallization of Amorphous Lactose in Nanocomposites Formed by Spray-Drying
    Öppna denna publikation i ny flik eller fönster >>Inhibition of Recrystallization of Amorphous Lactose in Nanocomposites Formed by Spray-Drying
    2015 (Engelska)Ingår i: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 104, nr 11, s. 3760-3769Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    This study aims at investigating the recrystallization of amorphous lactose in nanocomposites. In particular, the focus is on the influence of the nano- to micrometer length scale nanofiller arrangement on the amorphous to crystalline transition. Further, the relative significance of formulation composition and manufacturing process parameters for the properties of the nanocomposite was investigated. Nanocomposites of amorphous lactose and fumed silica were produced by co-spray-drying. Solid-state transformation of the lactose was studied at 43%, 84%, and 94% relative humidity using X-ray powder diffraction and microcalorimetry. Design of experiments was used to analyze spray-drying process parameters and nanocomposite composition as factors influencing the time to 50% recrystallization. The spray-drying process parameters showed no significant influence. However, the recrystallization of the lactose in the nanocomposites was affected by the composition (fraction silica). The recrystallization rate constant decreased as a function of silica content. The lowered recrystallization rate of the lactose in the nanocomposites could be explained by three mechanisms: (1) separation of the amorphous lactose into discrete compartments on a micrometer length scale (compartmentalization), (2) lowered molecular mobility caused by molecular interactions between the lactose molecules and the surface of the silica (rigidification), and/or (3) intraparticle confinement of the amorphous lactose.

    Nyckelord
    amorphous, crystallization, glass transition, mobility, physical stability, solid state, stabilization, spray drying, factorial design
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-266687 (URN)10.1002/jps.24583 (DOI)000362984100013 ()26182904 (PubMedID)
    Tillgänglig från: 2015-11-12 Skapad: 2015-11-10 Senast uppdaterad: 2018-01-10
    3. Structure and mobility of lactose in lactose/sodium montmorillonite nanocomposites
    Öppna denna publikation i ny flik eller fönster >>Structure and mobility of lactose in lactose/sodium montmorillonite nanocomposites
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    2016 (Engelska)Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 32, nr 49, s. 13214-13225Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    This study aims at investigating the molecular level organization and molecular mobility in montmorillonite nanocomposites with the uncharged organic low-molecular-weight compound lactose commonly used in pharmaceutical drug delivery, food technology, and flavoring. Nanocomposites were prepared under slow and fast drying conditions, attained by drying at ambient conditions and by spray-drying, respectively. A detailed structural investigation was performed with modulated differential scanning calorimetry, powder X-ray diffraction, solid-state nuclear magnetic resonance spectroscopy, scanning electron microscopy, microcalorimetry, and molecular dynamics simulations. The lactose was intercalated in the sodium montmorillonite interlayer space regardless of the clay content, drying rate, or humidity exposure. Although, the spray-drying resulted in higher proportion of intercalated lactose compared with the drying under ambient conditions, nonintercalated lactose was present at 20 wt % lactose content and above. This indicates limitations in maximum loading capacity of nonionic organic substances into the montmorillonite interlayer space. Furthermore, a fraction of the intercalated lactose in the co-spray-dried nanocomposites diffused out from the clay interlayer space upon humidity exposure. Also, the lactose in the nanocomposites demonstrated higher molecular mobility than that of neat amorphous lactose. This study provides a foundation for understanding functional properties of lactose/Na-MMT nanocomposites, such as loading capacity and physical stability.

    Nationell ämneskategori
    Materialkemi
    Identifikatorer
    urn:nbn:se:uu:diva-300158 (URN)10.1021/acs.langmuir.6b01967 (DOI)000389866300029 ()
    Tillgänglig från: 2016-08-05 Skapad: 2016-08-03 Senast uppdaterad: 2017-11-28
    4. Confinement of Amorphous Lactose in Pores formed upon Co-Spray-Drying with Nanoparticles
    Öppna denna publikation i ny flik eller fönster >>Confinement of Amorphous Lactose in Pores formed upon Co-Spray-Drying with Nanoparticles
    2017 (Engelska)Ingår i: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 106, nr 1, s. 322-330Artikel i tidskrift (Övrigt vetenskapligt) Published
    Abstract [en]

    This study aims at investigating factors influencing humidity induced recrystallization of amorphous lactose, produced by co-spray-drying with particles of cellulose nanocrystals (CNC) or sodium montmorillonite (Na-MMT). In particular, the focus is on how the nanoparticle shape and surface properties influence the nano- to micrometer length scale nanofiller arrangement in the nanocomposites and how the arrangements influence the mechanisms involved in the inhibition of the amorphous to crystalline transition. The nanocomposites were produced by co-spray-drying. Solid-state transformations were analyzed at 60-94% relative humidity using X-ray powder diffraction, microcalorimetry, and light microscopy. The recrystallization rate constant for the lactose/CNC and lactose/Na-MMT nanocomposites was lowered at nanofiller contents higher than 60% and were stable for months at 80% nanofiller. The most likely explanation to these results is spontaneous formations of mesoporous particle networks that the lactose is confined within upon co-spray-drying at high filler content. Compartmentalization and rigidification of the amorphous lactose proved to be less important mechanisms involved in the stabilization of lactose in the nanocomposites.

    Nationell ämneskategori
    Materialkemi
    Identifikatorer
    urn:nbn:se:uu:diva-300153 (URN)10.1016/j.xphs.2016.09.032 (DOI)000393920500035 ()27836110 (PubMedID)
    Tillgänglig från: 2016-08-03 Skapad: 2016-08-03 Senast uppdaterad: 2017-04-18Bibliografiskt granskad
    5. Humidity sorption of lactose/sodium montmorillonite nanocomposites
    Öppna denna publikation i ny flik eller fönster >>Humidity sorption of lactose/sodium montmorillonite nanocomposites
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Understanding of water sorption is of high importance in materials science as water may change materials properties promoting degradation, relaxations, or recrystallization. In this study, we investigated the humidity sorption in co-spray-dried lactose/sodium montmorillonite nanocomposites with varying lactose loading with the aim to increase the knowledge of the water sorption in this type of materials. It was demonstrated that the intercalation of lactose in the Na‑MMT clay decreased hygroscopicity of the composite despite high water affinity of both materials. As the cations in interlayer space of montmorillonite play an essential role in water sorption in the clay, we gained the molecular level understanding of Na+ interactions with lactose molecules and clay surface in the nanocomposites with molecular dynamic simulations and 23Na solid-state NMR. In conclusion, we demonstrated that the decreased hygroscopicity of the materials can be explained by interactions of lactose with the Na+ and the clay surfaces in the MMT interlayer space of lactose/Na-MMT nanocomposites.

    Nationell ämneskategori
    Materialkemi
    Identifikatorer
    urn:nbn:se:uu:diva-300154 (URN)
    Tillgänglig från: 2016-08-03 Skapad: 2016-08-03 Senast uppdaterad: 2016-09-01
    6. Powder compression mechanics of spray-dried lactose nanocomposites
    Öppna denna publikation i ny flik eller fönster >>Powder compression mechanics of spray-dried lactose nanocomposites
    2017 (Engelska)Ingår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 518, nr 1-2, s. 1-10Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The aim of this study was to investigate the structural impact of the nanofiller incorporation on the powder compression mechanics of spray-dried lactose. The lactose was co-spray-dried with three different nanofillers, that is, cellulose nanocrystals, sodium montmorillonite, and fumed silica, which led to lower micron sized nanocomposite particles with varying structure and morphology. The powder compression mechanics of the nanocomposites and physical mixtures of the neat spray-dried components were evaluated by a rational evaluation method with compression analysis as a tool using the Kawakita equation and the Shapiro-Konopicky-Heckel equation. Particle rearrangement dominated the initial compression profiles due to the small particle sizes of the materials. The strong contribution of particle rearrangement in the materials with fumed silica continued throughout the whole compression profile, which prohibited an in-depth material characterization. However, the lactose/cellulose nanocrystals and the lactose/sodium montmorillonite nanocomposites demonstrated increased yield pressure compared with the physical mixtures indicating increased particle hardness. This increase has likely to do with a reinforcement of the nanocomposite particles by skeleton formation of the nanoparticles. In summary, the rational evaluation applying compression analysis proved to be a valuable tool for mechanical evaluation for this type of materials unless they demonstrate particle rearrangement throughout the whole compression profile.

    Nyckelord
    Amorphous, Nanocomposite, Powder compression, Lactose, Spray-drying, Yield pressure
    Nationell ämneskategori
    Materialkemi
    Identifikatorer
    urn:nbn:se:uu:diva-300156 (URN)10.1016/j.ijpharm.2016.12.041 (DOI)000394402100001 ()28007544 (PubMedID)
    Tillgänglig från: 2016-08-05 Skapad: 2016-08-03 Senast uppdaterad: 2018-08-20
  • 11.
    Lilienberg, Elsa
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Biopharmaceutical Evaluation of Intra-arterial Drug-Delivery Systems for Liver Cancer: Investigations in healthy pigs and liver cancer patients2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    There are currently two types of intra-arterial drug-delivery system (DDS) in clinical use in the palliative treatment of primary liver cancer. The chemotherapeutic drug doxorubicin (DOX) can be formulated into a drug-in-lipiodol emulsion (LIPDOX) or a microparticulate drug-eluting bead system (DEBDOX). To facilitate development of future DDSs, we need to understand the release and local distribution of drug from these DDSs into the complex, in vivo, pathological environment.

    The overall aim of this project was to assess and improve understanding of the in vivo release of DOX from LIPDOX and DEBDOX and its local disposition in the liver. These processes were investigated in detail in a multisampling-site, healthy pig model and in human patients with liver cancer. The mechanisms involved in DOX disposition were studied by examining potential interactions between DOX and lipiodol and/or cyclosporine A (CsA) in pigs.  

    In this project, the main elimination pathway for DOX and its primary metabolite doxorubicinol (DOXol) was via bile; their extensive canalicular carrier-mediated transport (e.g. ATP-binding cassette transporters ABCB1, ABCC1, ABCC2 and ABCG2) was inhibited by CsA. CsA had no effect on the carbonyl and aldo-keto reductases responsible for the metabolism of DOX into DOXol. LIPDOX released DOX more rapidly and to a greater extent into the circulation than DEBDOX, which had only released 15% of the dose in patients after 24 hrs. The systemic exposure to DOX was lower for DEBDOX than for LIPDOX. Greater fractions of DOXol were formed in blood and bile with LIPDOX than with DEBDOX. This may have been because DOX was more widely distributed into regions with increased metabolic capacity or because of increased intracellular uptake when DOX was delivered in LIPDOX. The excipient lipiodol in the LIPDOX formulation did not interact with transporters, enzymes or membranes that would explain the increased cellular uptake of DOX.

    In conclusion, the release of DOX from DEBDOX is more controlled in vivo than that from LIPDOX, indicating that DEBDOX is a more robust pharmaceutical product. The formulations for future optimized DDSs should therefore be more similar to DEBDOX than to LIPDOX. 

    Delarbeten
    1. Investigation of Hepatobiliary Disposition of Doxorubicin Following Intrahepatic Delivery of Different Dosage Forms
    Öppna denna publikation i ny flik eller fönster >>Investigation of Hepatobiliary Disposition of Doxorubicin Following Intrahepatic Delivery of Different Dosage Forms
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    2014 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 11, nr 1, s. 131-144Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Unresectable, intermediate stage hepatocellular carcinoma (HCC) is often treated palliatively in humans by doxorubicin (DOX). The drug is administered either as a drug-emulsified-in-Lipiodol (DLIP) or as drug loaded into drug eluting beads (DEB), and both formulations are administered intrahepatically. However, several aspects of their in vivo performance in the liver are still not well-understood. In this study, DLIP and DEB were investigated regarding the local and systemic pharmacokinetics (PK) of DOX and its primary metabolite doxorubicinol (DOXol). An advanced PK-multisampling site acute in vivo pig model was used for simultaneous sampling in the portal, hepatic, and femoral veins and the bile duct. The study had a randomized, parallel design with four treatment groups (TI–TIV). TI (n = 4) was used as control and received an intravenous (i.v.) infusion of DOX as a solution. TII and TIII were given a local injection in the hepatic artery with DLIP (n = 4) or DEB (n = 4), respectively. TIV (n = 2) received local injections of DLIP in the hepatic artery and bile duct simultaneously. All samples were analyzed for concentrations of DOX and DOXol with UPLC-MS/MS. Compared to DLIP, the systemic exposure for DOX with DEB was reduced (p < 0.05), in agreement with a slower in vivo release. The approximated intracellular bioavailability of DOX during 6 h appeared to be lower for DEB than DLIP. Following i.v. infusion (55 min), DOX had a liver extraction of 41 (28–53)%, and the fraction of the dose eliminated in bile of DOX and DOXol was 20 (15–22)% and 4.2 (3.2–5.2)%, respectively. The AUCbile/AUCVP for DOX and DOXol was 640 (580–660) and 5000 (3900–5400), respectively. In conclusion, DLIP might initially deliver a higher hepatocellular concentration of DOX than DEB as a consequence of its higher in vivo release rate. Thus, DLIP delivery results in higher intracellular peak concentrations that might correlate with better anticancer effects, but also higher systemic drug exposure and safety issues.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-210381 (URN)10.1021/mp4002574 (DOI)000329529700012 ()24171458 (PubMedID)
    Tillgänglig från: 2013-11-06 Skapad: 2013-11-06 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
    2. The Effects of Lipiodol and Cyclosporin A on the Hepatobiliary Disposition of Doxorubicin in Pigs
    Öppna denna publikation i ny flik eller fönster >>The Effects of Lipiodol and Cyclosporin A on the Hepatobiliary Disposition of Doxorubicin in Pigs
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    2014 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 11, nr 4, s. 1301-1313Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Doxorubicin (DOX) emulsified in Lipiodol (LIP) is used as local palliative treatment for unresectable intermediate stage hepatocellular carcinoma. The objective of this study was to examine the poorly understood effects of the main excipient in the drug delivery system, LIP, alone or together with cyclosporin A (CsA), on the in vivo liver disposition of DOX. The advanced, multi-sampling-site, acute pig model was used; samples were collected from three blood vessels (v. portae, v. hepatica and v. femoralis), bile and urine. The four treatment groups (TI-TIV) all received two intravenous 5 min infusions of DOX into an ear vein: at 0 and 200 min. Before the second dose, the pigs received a portal vein infusion of saline (TI), LIP (TII), CsA (TIII) or LIP and CsA (TIV). Concentrations of DOX and its active metabolite doxorubicinol (DOXol) were analyzed using UPLC-MS/MS. A multi-compartment model was developed to describe the distribution of DOX and DOXol in plasma, bile and urine. LIP did not affect the pharmacokinetics of DOX or DOXol. CsA (TIII and TIV) had no effect on the plasma pharmacokinetics of DOX, but a 2-fold increase in exposure to DOXol and a significant decrease in hepatobiliary clearance of DOX and DOXol was observed. Model simulations supported that CsA inhibits 99% of canalicular biliary secretion of both DOX and DOXol, but does not affect the metabolism of DOX to DOXol. In conclusion, LIP did not interact with transporters, enzymes and/or biological membranes important for the hepatobiliary disposition of DOX.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-222282 (URN)10.1021/mp4007612 (DOI)000334092700022 ()24558959 (PubMedID)
    Tillgänglig från: 2014-04-09 Skapad: 2014-04-09 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
    3. Lipiodol does not affect the tissue distribution of intravenous doxorubicin infusion in pigs
    Öppna denna publikation i ny flik eller fönster >>Lipiodol does not affect the tissue distribution of intravenous doxorubicin infusion in pigs
    (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392Artikel i tidskrift (Övrigt vetenskapligt) Submitted
    Abstract
    Ort, förlag, år, upplaga, sidor
    Uppsala:
    Nyckelord
    doxorubicin, doxorubicinol, lipiodol, tissue distribution, Kp, cyclosporine A
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Farmaceutisk vetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-267119 (URN)
    Tillgänglig från: 2015-11-21 Skapad: 2015-11-18 Senast uppdaterad: 2018-01-10
    4. Comparison of drug release, pharmacokinetics and pharmacodynamics after lipiodol-based emulsion or drug-eluting bead delivery to patients with hepatocellular carcinoma
    Öppna denna publikation i ny flik eller fönster >>Comparison of drug release, pharmacokinetics and pharmacodynamics after lipiodol-based emulsion or drug-eluting bead delivery to patients with hepatocellular carcinoma
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nyckelord
    Doxorubicin, doxorubicinol, drug eluting beads, focal delivery, focal therapy, hepatocellular carcinoma, liver cancer, lipiodol, transarterial chemoembolization, transarterial infusion chemotherapy
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Farmaceutisk vetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-267395 (URN)
    Forskningsfinansiär
    Vetenskapsrådet, 521-2011-373
    Tillgänglig från: 2015-11-21 Skapad: 2015-11-21 Senast uppdaterad: 2018-01-10
  • 12.
    Vildhede, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    In vitro and in silico Predictions of Hepatic Transporter-Mediated Drug Clearance and Drug-Drug Interactions in vivo2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The liver is the major detoxifying organ, clearing the blood from drugs and other xenobiotics. The extent of hepatic clearance (CL) determines drug exposure and hence, the efficacy and toxicity associated with exposure. Drug-drug interactions (DDIs) that alter the hepatic CL may cause more or less severe outcomes, such as adverse drug reactions. Accurate predictions of drug CL and DDI risk from in vitro data are therefore crucial in drug development.

    Liver CL depends on several factors including the activities of transporters involved in the hepatic uptake and efflux. The work in this thesis aimed at developing new in vitro and in silico methods to predict hepatic transporter-mediated CL and DDIs in vivo. Particular emphasis was placed on interactions involving the hepatic uptake transporters OATP1B1, OATP1B3, and OATP2B1. These transporters regulate the plasma concentration-time profiles of many drugs including statins.

    Inhibition of OATP-mediated transport by 225 structurally diverse drugs was investigated in vitro. Several novel inhibitors were identified. The data was used to develop in silico models that could predict OATP inhibitors from molecular structure. Models were developed for static and dynamic predictions of in vivo transporter-mediated drug CL and DDIs. These models rely on a combination of in vitro studies of transport function and mass spectrometry-based quantification of protein expression in the in vitro models and liver tissue. By providing estimations of transporter contributions to the overall hepatic uptake/efflux, the method is expected to improve predictions of transporter-mediated DDIs. Furthermore, proteins of importance for hepatic CL were quantified in liver tissue and isolated hepatocytes. The isolation of hepatocytes from liver tissue was found to be associated with oxidative stress and degradation of transporters and other proteins expressed in the plasma membrane. This has implications for the use of primary hepatocytes as an in vitro model of the liver. Nevertheless, by taking the altered transporter abundance into account using the method developed herein, transport function in hepatocyte experiments can be scaled to the in vivo situation. The concept of protein expression-dependent in vitro-in vivo extrapolations was illustrated using atorvastatin and pitavastatin as model drugs.

    Delarbeten
    1. Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs): Influence of Protein Expression on Drug - Drug Interactions
    Öppna denna publikation i ny flik eller fönster >>Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs): Influence of Protein Expression on Drug - Drug Interactions
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    2012 (Engelska)Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, nr 10, s. 4740-4763Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The hepatic organic anion transporting polypeptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug-drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1, The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-176227 (URN)10.1021/jm300212s (DOI)000304338800017 ()
    Tillgänglig från: 2012-06-19 Skapad: 2012-06-18 Senast uppdaterad: 2017-12-07Bibliografiskt granskad
    2. Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions
    Öppna denna publikation i ny flik eller fönster >>Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions
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    2014 (Engelska)Ingår i: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, nr 7, s. 1210-1218Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Differences in the expression and function of the organic anion transporting polypeptide (OATP) transporters contribute to interindividual variability in atorvastatin clearance. However, the importance of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) in atorvastatin uptake clearance (CLupt) is not yet clarified. To elucidate this issue, we investigated the relative contribution of NTCP, OATP1B1, OATP1B3, and OATP2B1 to atorvastatin CLupt in 12 human liver samples. The impact of inhibition on atorvastatin CLupt was also studied, using inhibitors of different isoform specificities. Expression levels of the four transport proteins were quantified by liquid chromatography tandem mass spectrometry. These data, together with atorvastatin in vitro kinetics, were used to predict the maximal transport activity (MTA) and interindividual differences in CLupt of each transporter in vivo. Subsequently, hepatic uptake impairment on coadministration of five clinically interacting drugs was predicted using in vitro inhibitory potencies. NTCP and OATP protein expression varied 3.7- to 32-fold among the 12 sample donors. The rank order in expression was OATP1B1 > OATP1B3 approximate to NTCP approximate to OATP2B1. NTCP was found to be of minor importance in atorvastatin disposition. Instead, OATP1B1 and OATP1B3 were confirmed as the major atorvastatin uptake transporters. The average contribution to atorvastatin uptake was OATP1B1 > OATP1B3 >> OATP2B1 > NTCP, although this rank order varied among individuals. The interindividual differences in transporter expression and CLupt resulted in marked differences in drug-drug interactions due to isoform-specific inhibition. We conclude that this variation should be considered in in vitro to in vivo extrapolations.

    Nationell ämneskategori
    Farmakologi och toxikologi Medicinska och farmaceutiska grundvetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-229430 (URN)10.1124/dmd.113.056309 (DOI)000338341300016 ()
    Tillgänglig från: 2014-08-08 Skapad: 2014-08-07 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
    3. Comparative proteomics of human liver tissue and hepatocytes reveal systematic differences in proteins determining hepatic drug exposure
    Öppna denna publikation i ny flik eller fönster >>Comparative proteomics of human liver tissue and hepatocytes reveal systematic differences in proteins determining hepatic drug exposure
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    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nyckelord
    Quantitative proteomics, Oxidative stress, plasma membrane protein, drug transport and metabolism, pitavastatin, Total Protein Approach, ADME, OATP, CYP, UGT
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Biofarmaci
    Identifikatorer
    urn:nbn:se:uu:diva-241374 (URN)
    Tillgänglig från: 2015-01-12 Skapad: 2015-01-12 Senast uppdaterad: 2018-01-11
    4. Mechanistic Modeling of Pitavastatin Disposition in Sandwich-Cultured Human Hepatocytes: A Proteomics-Informed Bottom-Up Approach
    Öppna denna publikation i ny flik eller fönster >>Mechanistic Modeling of Pitavastatin Disposition in Sandwich-Cultured Human Hepatocytes: A Proteomics-Informed Bottom-Up Approach
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    2016 (Engelska)Ingår i: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 44, nr 4, s. 505-516Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Isolated human hepatocytes are commonly used to predict transporter-mediated clearance in vivo. Such predictions, however, do not provide estimations of transporter contributions and consequently do not allow predictions of the outcome resulting from a change in the activity of a certain transporter, e.g., by inhibition or a genetic variant with reduced function. Pitavastatin is a drug that is heavily dependent on hepatic transporters for its elimination and it is mainly excreted as unchanged drug in the bile. For this reason, pitavastatin was used as a model drug to demonstrate the applicability of a bottom-up approach to predict transporter-mediated disposition in sandwich-cultured human hepatocytes (SCHH), allowing for the estimation of transporter contributions. Transport experiments in transfected HEK293 cells and inverted membrane vesicles overexpressing each of the relevant transport proteins were used to generate parameter estimates for the mechanistic model. By adjusting for differences in transporter abundance between the in vitro systems and individual SCHH batches, the model successfully predicted time profiles of medium and intracellular accumulation. Our predictions of pitavastatin bile accumulation could, however, not be confirmed due to a very low biliary excretion of pitavastatin in relation to the hepatic uptake in our SCHH. This study is, to our knowledge, the first to successfully simulate transporter-mediated processes in a complex system such as SCHH at the level of individual transport proteins using a bottom-up approach.

    Nyckelord
    pitavastatin, SCHH, hepatocytes, drug transport, OATP, NTCP, hepatic uptake, Pgp, BCRP, MRP2, MRP3
    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Forskningsämne
    Biofarmaci
    Identifikatorer
    urn:nbn:se:uu:diva-241375 (URN)10.1124/dmd.115.066746 (DOI)000372880600005 ()26842596 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet, 2822Stiftelsen Lars Hiertas Minne
    Tillgänglig från: 2015-01-12 Skapad: 2015-01-12 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
  • 13.
    Widenbring, Ronja
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Microgel Interactions with Peptides and Proteins: Consequence of Peptide and Microgel Properties2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Microgels are lightly cross-linked hydrogel particles in the sub-micrometer to micrometer size range with a capacity to drastically change their volume in response to changes in the external environment. Microgels have an ability to bind and store substances such as biomacromolecular drugs, notably proteins and peptides, and release them upon stimuli, making them potential candidates as drug delivery vehicles and functional biomaterials. This thesis aims at clarifying important factors affecting peptide-microgel interactions. These interactions were studied by micromanipulator-assisted light and fluorescence microscopy focusing on microgel deswelling in response to peptide binding, as well as re-swelling in response to peptide release or enzymatic degradation. To evaluate peptide uptake in microgels, solution depletion measurements were used whereas the peptide secondary structure was investigated by circular dichroism. In addition, the peptide and enzyme distribution within microgels was analyzed with confocal microscopy.

    Results presented in this thesis demonstrate that peptide incorporation into microgels, as well as peptide-induced microgel deswelling, increases with peptide length and charge density. In addition, results demonstrate that the peptide charge (length) rather than peptide charge density determines microgels deswelling. End-to-end cyclization is shown to not noticeably influence peptide-microgel interactions, suggesting that peptide cyclization can be used in combination with oppositely charged microgel carriers to improve the proteolytic and chemical stability of the peptide compared to the corresponding linear variant. Peptide secondary structure is found to drastically affect peptide incorporation into, and release from, oppositely charged microgels. Furthermore, it is shown that microgel charge density, peptide molecular weight, and enzyme concentration all greatly influence microgel bound peptide degradation. Of importance for applications, protective effects of microgels against proteolytic peptide degradation are observed only at sufficiently high microgel charge densities. Enzyme-mediated microgel degradation is shown to increase with increasing enzyme concentration, while an increased peptide loading in microgels causes a concentration-dependent decrease in microgel degradation.

    Taken together, results obtained in this work have provided some insight into factors of importance for rational use of microgels as delivery systems for protein or peptide drugs, but also in a host of other biomedical applications using weakly cross-linked polymer systems.

    Delarbeten
    1. Effects of Peptide Secondary Structure on the Interaction with Oppositely Charged Microgels
    Öppna denna publikation i ny flik eller fönster >>Effects of Peptide Secondary Structure on the Interaction with Oppositely Charged Microgels
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    2011 (Engelska)Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, nr 2, s. 419-424Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The importance of peptide secondary structure on the interaction between antimicrobial peptides and oppositely charged poly(acrylic acid-co-acrylamide) microgels of various charge density was investigated for EFKRIVQRIKDFLRNLV (EFK17). Through D-enantiomer (EFK17-d/a; E(dF)KR(dI)VQR(dI)KD(dF)LRNLV) or tryptophan (EFK17-W/a; EWKRWVQRWKDFLRNLV) substitutions, both conformation-dependent and -independent amphiphilicity of this peptide could be precisely controlled. Peptide secondary structure was investigated by circular dichroism, whereas microgel deswelling and reswelling in response to peptide binding and release were studied by micromanipulator-assisted light and fluorescence microscopy, and peptide uptake in the microgels was determined from solution depletion measurements. Results show that peptide binding to the microgel is highly influenced by peptide secondary structure. EFK17-a, characterized by an idealized helix with all polar/charged amino acids located at one side of the helix, and all nonpolar/hydrophobic residues on the other, displays pronounced alpha-helix induction on peptide binding to the microgels. EFK17-d/a, on the other hand, displays no such amphiphilic helix induction. Mirroring this, EFK17-a displays substantially higher binding to the microgels than EFK17-d/a as well as much larger peptide-induced microgel deswelling. For EFK17-W/a, both conformation-dependent and -independent amphiphilicity effects were demonstrated. Overall, the results show that peptide conformational aspects need to be considered in peptide/microgel interactions, for example, in the design of microgel carrier systems for peptide drugs.

    Nationell ämneskategori
    Farmaceutiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-149054 (URN)10.1021/bm101165e (DOI)000287175700017 ()21182237 (PubMedID)
    Tillgänglig från: 2011-03-15 Skapad: 2011-03-15 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
    2. Effects of peptide cyclization on the interaction with oppositely charged microgels
    Öppna denna publikation i ny flik eller fönster >>Effects of peptide cyclization on the interaction with oppositely charged microgels
    2011 (Engelska)Ingår i: Colloids and Surfaces A: Physicochemical and Engineering Aspects, ISSN 0927-7757, E-ISSN 1873-4359, Vol. 391, nr 1-3, s. 62-68Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The effect of peptide cyclization on the interaction between antimicrobial peptides and oppositely charged poly(acrylic acid-co-acrylamide) microgels of various charge density was investigated for linear and cyclic variants of peptide oligomers (C(ARKKAAKA)nC) (n = 1, 1.5, 2, 3). Through this, peptide length could be varied without substantially affecting peptide charge density and mean hydrophobicity. Furthermore, the peptides were demonstrated to display random coil conformation both in aqueous solution and when bound to oppositely charged microgels, allowing effects of cyclization to be monitored without interference from conformational transitions. With increasing peptide length, both cyclic and linear peptide variants displayed increased binding affinity to oppositely charged microgels. For all peptide lengths, however, the difference between cyclic and linear peptide variants was marginal at most, hence cyclization had little or no influence in peptide incorporation to oppositely charged microgels. In parallel, microgel deswelling increased with peptide length for both linear and cyclic peptide variants, while linear and cyclic peptide variants of the same length displayed very similar peptide-induced deswelling. Also electrolyte-induced peptide desorption from the microgels was similar for linear and cyclic peptide variants. Taken together, these findings demonstrate that end-to-end cyclization does not markedly affect peptide incorporation into, and release from, oppositely charged microgels. This opens up opportunities for the use of microgels as carriers for peptides which have been cyclized in order to improve their proteolytic and chemical stability, or in order to achieve other therapeutic advantages compared to the corresponding linear peptide variant.

    Nyckelord
    Cyclization, Microgel, Peptide
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-168831 (URN)10.1016/j.colsurfa.2011.01.029 (DOI)000299068100009 ()
    Anmärkning
    18th International Symposium on Surfactants in Solution (SIS), Melbourne Australia, 14-19 November 2010Tillgänglig från: 2012-02-16 Skapad: 2012-02-16 Senast uppdaterad: 2017-12-07Bibliografiskt granskad
    3. Peptide-Microgel Interactions in the Strong Coupling Regime
    Öppna denna publikation i ny flik eller fönster >>Peptide-Microgel Interactions in the Strong Coupling Regime
    2012 (Engelska)Ingår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, nr 35, s. 10964-10975Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The interaction between lightly cross-linked poly(acrylic acid) microgels and oppositely charged peptides was investigated as a function of peptide length, charge density, pH, and salt concentration, with emphasis on the strong coupling regime at high charge contrast. By micromanipulator-assisted light microscopy, the equilibrium volume response of single microgel particles upon oligolysine and oligo(lysine/alanine) absorption could be monitored in a controlled fashion. Results show that microgel deswelling, caused by peptide binding and network neutralization, increases with peptide length (3 < 5 < 10) and charge density (30% < 50% < 100%). Furthermore, oligomer-induced microgel deswelling was more pronounced at pH 5 than at pH 8, reflecting the lower network charge density in the former case (pK(a) for the isolated acrylic acid approximate to 4.7). In order to describe these highly coupled systems, a model was developed, in which counterion/peptide-mediated electrostatic attraction between the network chains is described using an exponential force law, and the network elasticity by the inverse Langevin theory. The model was used to calculate the composition of microgels in contact with reservoir solutions of peptides and simple electrolytes. At high electrostatic coupling, the calculated swelling curves were found to display first-order phase transition behavior. The model was demonstrated to capture pH- and electrolyte-dependent microgel swelling, as well as effects of peptide length and charge density on microgel deswelling. The analysis demonstrated that the peptide charge (length), rather than the peptide charge density, determines microgel deswelling. Furthermore, a transition between continuous and discrete network collapse was identified, consistent with experimental results in the present investigations, as well as with results from the literature on microgel deswelling caused by multivalent cations.

    Nationell ämneskategori
    Kemi
    Identifikatorer
    urn:nbn:se:uu:diva-182754 (URN)10.1021/jp306121h (DOI)000308339400060 ()
    Tillgänglig från: 2012-10-18 Skapad: 2012-10-15 Senast uppdaterad: 2017-12-07Bibliografiskt granskad
    4. Factors Affecting Enzymatic Degradation of Microgel-Bound Peptides
    Öppna denna publikation i ny flik eller fönster >>Factors Affecting Enzymatic Degradation of Microgel-Bound Peptides
    2013 (Engelska)Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, nr 7, s. 2317-2325Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Proteolytic degradation and release of microgel-bound peptides was investigated for trypsin, poly(acrylic acid-co-acrylamide) microgels (70-90 mu m in diameter), and oppositely charged polylysine, using a method combination of confocal microscopy and micromanipulator-assisted light microscopy. Results show that trypsin-induced release of polylysine increased with increasing trypsin concentration, decreasing microgel charge density and decreasing peptide molecular weight. While the microgel offered good protection against enzymatic degradation at high microgel charge density, it was also observed that the cationic peptide enabled trypsin to bind throughout the peptide-loaded microgels, even when it did not bind to the peptide-void ones. With the exception of highly charged microgels, proteolytic degradation throughout the peptide-loaded microgel resulted in the generation of short and non-adsorbing peptide stretches, giving rise to the concentration and peptide length dependence observed. A simple random scission model was able to qualitatively capture these experimental findings. collectively, the results demonstrate that microgel charge density, peptide molecular weight, and enzyme concentration greatly influence degradation/release of microgel-bound peptides and need to be considered in the use of microgels, e.g., as carriers for protein and peptide drugs.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-204977 (URN)10.1021/bm400431f (DOI)000321793700021 ()
    Tillgänglig från: 2013-08-16 Skapad: 2013-08-13 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
    5. Chain and Pore-Blocking Effects on Matrix Degradation in Protein-Loaded Microgels
    Öppna denna publikation i ny flik eller fönster >>Chain and Pore-Blocking Effects on Matrix Degradation in Protein-Loaded Microgels
    2014 (Engelska)Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, nr 10, s. 3671-3678Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Factors affecting matrix degradation in protein-loaded microgels were investigated for dextran-based microgels, the sugar-binding protein Concanavalin A (ConA), and the dextran-degrading enzyme Dextranase. For this system, effects of enzyme, protein, and glucose concentrations, as well as pH, were considered. Microgel network degradation was monitored by micromanipulator-assisted light microscopy, whereas enzyme and protein distributions were monitored by confocal microscopy. Results show that Dextranase-mediated microgel degradation increased with increasing enzyme concentration, whereas an increased ConA loading in the dextran microgels caused a concentration-dependent decrease in microgel degradation. In the presence of glucose, competitive release of microgel-bound ConA restored the microgel degradation observed in the absence of ConA. To clarify effects of mass transport limitations, microgel degradation was compared to that of non-cross-linked dextran, demonstrating that ConA limits enzyme substrate access in dextran microgels primarily through pore blocking and induction of pore shrinkage. The experimentally observed effects were qualitatively captured by a modified Michaelis-Menten approach for spherical symmetry, in which network blocking by ConA was included. Taken together, the results demonstrate that matrix degradation of protein-loaded microgels depends sensitively on a number of factors, which need to be considered in the use of microgels in biomedical applications.

    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-236549 (URN)10.1021/bm5009525 (DOI)000343026600022 ()25144139 (PubMedID)
    Tillgänglig från: 2014-11-26<