16 research outputs found
Immobilisoitujen entsyymimikroreaktorien kehittäminen sytokromi P450 -välitteisen lääkeainemetabolian tutkimiseen
The cytochrome P450 (CYP) enzyme family is responsible for eliminating exogenous chemicals from the human body. As most drugs are metabolized by the same small number of CYP isoenzymes, the risk of drug-drug interactions grows as the use of drugs increases. Consequently, it is important to study the metabolic pathways of a new drug candidate as early in the development pipeline as possible to be able to either modify the molecule or abandon the candidate before continuing to the cost-intensive clinical stage.
The general aim of this thesis was to develop new immobilization methods for cytochrome P450 enzymes with a view to implementation of immobilized enzyme microreactors for pharmaceutical applications. Two main approaches for immobilizing CYP-containing human liver microsomes (HLM) were studied: the immobilization of HLM on commercial streptavidin-coated magnetic particles and the immobilization of HLM on in-house fabricated thiol-ene based microfluidic chips. On the basis of literature search, a novel immobilization method utilizing biotin-labelled fusogenic liposomes (FL) was developed.
When immobilizing HLM on magnetic particles, the use of FL conferred a 3-fold increase in enzyme activity compared to a previously published method based on physical adsorption on the bead surface. The Km value of the immobilized HLM was determined, and was comparable to the Km of the soluble HLM (2.5 ± 0.49 μM and 0.5-2 μM, respectively), which is essential for the prospective applications in metabolic studies. Enzyme stability remains an issue, as the activity of the immobilized enzyme quickly decreased with consecutive incubations, probably due to both thermal inactivation and leaching.
Three different methods for HLM immobilization on thiol-ene micropillar chips were studied. HLM were solubilized on a chip surface functionalized with lipid bilayers and HLM labeled with biotin using FL was bound on streptavidin-functionalized chip surface. For comparison, HLM was also immobilized on chip surface by physical adsorption. The highest initial activities could be achieved by adsorption, but the activity also decreased very rapidly. With the other two methods, initial reactor activities were lower, but the decline of enzyme activity could be slowed down. However, the enzyme activity of all reactors faded within 2 hours.
In the future, practical applications based on the developed immobilization approaches could be developed for in vitro studies of human drug metabolism. Another valid application for these reactors is the in situ preparation of analytical standards of CYP metabolites.Sytokromi P450 (CYP) entsyymiperhe vastaa vierasaineiden poistamisesta ihmiskehosta. Koska useimpien lääkeaineiden metaboliaa katalysoivat samat CYP-isoentsyymit, lääkeaineinteraktioiden riski kasvaa, kun lääkkeiden käyttö yhteiskunnassa lisääntyy. Uusien lääkeaineiden metaboliareittejä onkin tärkeää kartoittaa jo varhaisessa vaiheessa lääkekehitystä, jotta molekyyliä voidaan tarvittaessa muokata tai kehityslinja hylätä kokonaan ennen kallista kliinistä vaihetta.
Tämän työn tavoitteena oli kehittää uusia immobilisointimenetelmiä CYP-entsyymeille farmaseuttisia sovelluksia varten. Työssä tutkittiin kahta menetelmää CYP-entsyymejä sisältävien ihmisen maksamikrosomien (human liver microsomes, HLM) immobilisointiin. Entsyymejä immobilisoitiin sekä kaupallisten, streptavidiinipäällysteisten magneettipartikkeleiden pintaan että itse valmistettujen tioleenipohjaisten mikrosirujen pintaan. Tarkoitukseen kehitettiin kirjallisuushaun perusteella uusi, biotiinileimattuja fusogeenisiä liposomeja hyödyntävä immobilisointimenetelmä.
Fusogeenisten liposomien hyödyntäminen nosti magneettipartikkelien pintaan immobilisoitujen entsyymien entsyymiaktiivisuutta kolminkertaisesti verrattuna aikaisemmin raportoituun adsorptiopohjaiseen menetelmään. Immobilisoidulle HLM:lle määritetty Km-arvo (2.5 ± 0.49 μM) vastasi hyvin kirjallisuudessa liukoiselle HLM:lle raportoitua arvoa (0.5-2 μM), mikä on oleellista mahdollisten tulevien sovellusten kannalta. Immobilisoidun entsyymin aktiivisuus kuitenkin laski nopeasti perättäisten inkubointien seurauksena, mikä johtui todennäköisesti sekä entsyymin huuhtoutumisesta että lämmön aikaansaamasta inaktivaatiosta.
Tioleenipohjaisten mikrosirujen pintaan mikrosomeja immobilisoitiin kolmella menetelmällä. Mikrosomeja liuotettiin lipidi-kaksoiskerroksella funktionalisoidun kanavan pintaan ja biotiinilla leimattuja mikrosomeja immobilisoitiin streptavidiinilla funktionalisoidun sirun pintaan. Verrokkisirussa HLM immobilisoitiin sirun pintaan fysikaalisen adsorption avulla. Suurin alkuaktiivisuus saavutettiin adsorptiolla, mutta entsyymiaktiivisuus myös laski nopeasti. Kahta muuta menetelmää hyödyntämällä alkuaktiivisuudet olivat matalampia, mutta aktiivisuus myös tippui hitaammin. Kaikkien reaktorityyppien aktiivisuus hävisi kahden tunnin sisällä reaktion aloittamisesta.
Tulevaisuudessa työssä kehitettyjä immobilisointimenetelmiä voidaan käyttää pohjana suunniteltaessa käytännön sovelluksia ihmisen vierasainemetabolian tutkimiseen. Toinen mahdollinen sovelluskohde kehitetyille entsyymireaktoreille on analyyttisten metaboliittistandardien tuottaminen
Immobilized enzyme microreactors in drug metabolism research
Drug metabolism is an enzyme-catalyzed process that has major implications for a drug’s safety and efficacy. Consequently, evaluating the metabolic properties of a new drug candidate is of paramount importance already in the preclinical phase of drug development.
Although the available in vitro techniques for drug metabolism research have improved over recent years, the state-of-the-art methodology relies on enzyme assays conducted in static conditions, with limited spatiotemporal control of assay variables. Conducting metabolic assays under flow-through conditions could pave the way for more in-depth understanding of the mechanistic basis underlying drug-enzyme and drug-drug interactions. This, however, requires that drug-metabolizing enzymes be immobilized onto a solid support material without compromising protein folding or enzyme function. Although a wealth of different enzyme immobilization strategies are currently available, most of them are not amenable to immobilization of the microsomal, drug-metabolizing enzymes.
The aim of this thesis was to establish immobilized enzyme microreactor (IMER) platforms for studying drug metabolism under flow conditions, thereby improving the in vitro-in vivo prediction of the metabolic fate of new drug candidates. The use of microfluidics and microfabrication technology facilitated the straightforward implementation of flow-through assays and furthermore allows their multiplexing (parallelism) and integration with other operational units on a single platform, minimizing both reagent consumption and dead volumes.
In the first sub-project (publication I), a novel strategy for the immobilization of the membrane-bound enzymes cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) was designed and implemented on microreactors fabricated from off-stoichiometric thiol-enes (OSTE). The strategy was based on biotinylation of human liver microsomes via biotin-tagged fusogenic liposomes, and utilized the tunable surface chemistry of OSTEs to allow easy functionalization of the microreactor surface. The IMER platform was preliminarily characterized to ascertain enzyme stability and preservation of key enzyme kinetic parameters, with an emphasis on CYP-mediated (phase I) oxidoreductive reactions.
In the second sub-project (publication II), the feasibility of the IMER platform for studying the kinetics of UGT-mediated drug conjugation (phase II) was assessed, with an emphasis on the mechanistic basis for the pronounced underestimation of in vivo clearance kinetics by the currently available static in vitro techniques. In particular, the effect of membrane disruption and fatty-acid inhibition on UGT-kinetics in vitro was studied in detail. The kinetics of zidovudine glucuronidation (the model reaction used in this study) under flow-through conditions was shown to be in good agreement with that obtained using microsomal incubations in static conditions without the need for added pore-forming agents such as alamethicin.
In the third sub-project (publication III), the technology was further developed by incorporating a highly overlooked dimension in the assay design: the impact of oxygen partial pressure on the metabolic fate of drug candidates. This was achieved by exploiting the unique material-induced oxygen scavenging property of thiol-ene polymers. The developed chip design allowed the rapid and simple control of oxygen concentration in enzymatic assays, which is difficult to achieve with conventional static assay methods.
Overall, the developed methodology was shown to retain enzyme activity and native enzyme kinetic parameters of both CYP and UGT enzymes, an unconditional prerequisite for drug metabolism assays. With the immobilization method utilizing membrane biotinylation via liposome fusion, common problems (such as diffusion-limited kinetics and enzyme inactivation) associated with conventional immobilization approaches were circumvented. Furthermore, the universal applicability of the immobilization method for all membrane-bound enzymes was preliminarily demonstrated with recombinant CYP enzymes in sub-project/publication I.
The methodology developed here also enabled mechanistic studies focused on the alamethicin-induced membrane disruption (commonly used in UGT assays to overcome mass transfer limitations) and the proposed inhibitory effects of fatty acids, which may shed light on the foundations behind the poor in vivo correlation associated with UGT reactions in vitro.
The material-induced oxygen scavenging facilitated by OSTE polymers, together with microfluidic actuation, was shown to be an easily controllable approach for adjusting the oxygen partial pressure on demand. The advantage of this approach is that, unlike in typical approaches, complex chip designs or pressurized compartments are not needed. Beyond the demonstrated feasibility of the IMER platform for studying the impact of NADPH-cytochrome P450 oxidoreductase (POR)-mediated metabolism, the theoretical framework established here for OSTE-enabled oxygen control in microfluidic assays is likely to find many applications, particularly in organ-on-a-chip research.
In conclusion, the microreactor platform developed in this thesis offers an enabling toolbox for conducting in vitro drug metabolism assays under flow conditions that circumvents many of the key shortcomings of current state-of-the-art (static) in vitro enzyme assay methodology, as well as those of previously reported IMER platforms. At the time of publication, the methodology developed herein has already been utilized in several follow-up studies and has shown utility in diverse applications beyond this work.Lääkeainemetabolia on entsyymivälitteinen prosessi, jossa elimistö muokkaa lääkeaineita helpommin eritettävään muotoon. Lääkeainemetabolialla on suuri merkitys sekä lääkkeen turvallisuuden että tehon kannalta. Tästä syystä uuden lääkeaineen metaboliaa on ensiarvoisen tärkeää kartoittaa jo lääkekehityksen varhaisessa, prekliinisessä vaiheessa.
Vaikka lääkeainemetabolian tutkimiseen käytetyt in vitro -menetelmät ovatkin kehittyneet viime vuosina, on kokeellisten parametrien säätö ajan ja paikan suhteen haastavaa nykyisillä staattisilla kuoppalevymenetelmillä. Metaboliakokeiden suorittaminen virtausolosuhteissa voisi auttaa tutkijoita lääke-entsyymi- ja lääke-lääke-yhteisvaikutusten mekanismien ymmärtämisessä. Virtausolosuhteiden hyödyntämiseksi lääkeaineita metaboloivat entsyymit pitää kuitenkin kiinnittää eli immobilisoida kiinteään kantajamateriaaliin. Immobilisointi ei saisi kuitenkaan muuttaa entsyymin aktiivisuutta tai lääkkeen ja entsyymin välisten vuorovaikutusten voimakkuutta. Vaikka entsyymien immobilisointiin on tarjolla runsaasti eri menetelmiä, useimmat niistä soveltuvat huonosti solukalvoon sitoutuneille entsyymeille, joihin kuuluvat myös tärkeimmät lääkeaineita metaboloivat entsyymit, sytokromi P450 (CYP) ja UDP-glukuronosyylitransferaasi (UGT).
Tässä väitöskirjatyössä kehitettiin moderneja mikrovalmistusmenetelmiä hyödyntäen entsyymimikroreaktoreita lääkeainemetabolian tutkimiseen virtausolosuhteissa. Mikrofluidistiikkan hyödyntäminen mahdollisti kokeellisten parametrien, kuten lääkeainepitoisuuden, yksinkertaisen säädön kokeen aikana. Mikrovalmistettujen reaktorien liittäminen yhteen muiden toiminnallisten yksiköiden kanssa on myös helppoa, mikä puolestaan vähentää sekä reagenssien kulutusta että systeemin aikaviivettä eli ns. kuollutta tilavuutta.
Ensimmäisessä osajulkaisussa kehitettiin uudenlainen menetelmä solukalvoon sitoutuneiden, lääkeaineita metaboloivien entsyymien (CYP ja UGT) immobilisointiin. Tämä menetelmä perustui ihmisen maksamikrosomien biotinylointiin biotiinilla leimattujen, solukalvojen kanssa spontaanisti fuusioituvien liposomien avulla. Biotinyloidut maksamikrosomit immobilisoitiin tioleenipolymeereistä valmistettuihin, avidiinilla pinnoitettuihin mikroreaktoreihin. Tulosten perusteella maksamikrosomien immobilisointi ei vaikuttanut malliaineiden CYP-entsyymiaffiniteettiin.
Toisessa osajulkaisussa selvitettiin kehitetyn mikroreaktorikonseptin soveltuvuutta UGT-entsyymien kinetiikan tutkimiseen virtausolosuhteissa. Tyypillinen ongelma UGT-välitteisen metabolian tutkimuksessa on nykyisten in vitro -menetelmien huono in vivo -korrelaatio. Työssä pyrittiin tutkimaan ilmiön mekanistista taustaa virtausolosuhteita hyödyntämällä. Erityisesti tutkittiin solukalvon yli tapahtuvan aineensiirron ja rasvahappovälitteisen inhibition vaikutusta UGT-metaboliaan in vitro. UGT-metabolian malliaineena käytetyn tsidovudiinin kinetiikka virtausolosuhteissa vastasi hyvin staattisilla menetelmillä saatuja tuloksia, ilman tarvetta solukalvoa rikkoville lisäaineille, kuten alametisiinille.
Kolmannessa osajulkaisussa mikroreaktorikonseptia täydennettiin hapen osapaineen säädöllä. Tämä toteutettiin hyödyntämällä tioleenipolymeerin ainutlaatuista kykyä reagoida molekulaarisen hapen kanssa. Työssä osoitettiin, että tioleenien aikaansaaman happidepleetion avulla on mahdollista säätää hapen tasoa mikroreaktorin sisällä nopeammin ja yksinkertaisemmin kuin perinteisillä menetelmillä.
Työssä kehitetyt menetelmät säilyttivät tutkittujen CYP- ja UGT-entsyymien aktiivisuuden ja entsyymikineettiset ominaisuudet, mikä on ensisijaisen tärkeää käytännön sovellusten kannalta. Solukalvon biotinylointiin perustuva immobilisointimenetelmä ei kärsi tavanomaisten menetelmien tapaan diffuusiorajoitteisesta kinetiikasta tai entsyymi-inaktivaatiosta. Lisäksi menetelmä soveltuu minkä tahansa solukalvoproteiinin immobilisointiin.
Tioleenipolymeerien kyky reagoida hapen kanssa, yhdistettynä mikrofluidistiikan hyödyntämiseen mahdollistivat happipitoisuuden nopean säädön mikroreaktorin sisällä. Verrattuna aikaisempiin teknisiin ratkaisuihin, työssä kehitetty menetelmä hapen säätöön ei vaadi monimutkaisia rakenteita tai paineistettuja kaasuja. Työssä tsidovudiinilla osoitetun happiherkän lääkeainemetabolian tutkimisen lisäksi kehitetyllä alustalla on runsaasti sovelluksia esimerkiksi mikrofluidistiikkaa hyödyntävissä solukasvatuksissa (engl. organ-on-a-chip).
Yhteenvetona voidaan todeta, että kehitetty mikroreaktorikonsepti tarjoaa monipuolisen työkalun lääkeainemetabolian tutkimiseen virtausolosuhteissa, ja ratkaisee useita sekä nykyisiin staattisiin in vitro -malleihin että jo käytössä oleviin mikroreaktorimalleihin liittyviä haasteita ja ongelmia. Kehitetty immobilisointimenetelmä ei tavanomaisten menetelmien tapaan vaikuta kalvoon sitoutuneiden entsyymien stabiiliuteen tai entsyymiaffiniteettiin, ja soveltuu siten erityisesti CYP- ja UGT-välitteisen metabolian tutkimukseen. Läpivirtausreaktoreihin perustuva lääkeainemetaboliatutkimus taas mahdollistaa uudentyyppisiä koeasetelmia, joilla on sovelluksia erityisesti esimerkiksi lääke-entsyymi- ja lääke-lääke-yhteisvaikutusten mekanismien tutkimisessa
Understanding Cell Proliferation and Material induced Cell Death on Microfluidic Devices Made of Off Stoichiometric Thiol enes
Peer reviewe
Mechanistic study of oxygen-scavenging properties of off-stoichiometric thiol-enes
Peer reviewe
Microfluidic oxygen tolerability screening of nanocarriers for triplet fusion photon upconversion
The full potential of triplet fusion photon upconversion (TF-UC) of providing high-energy photons locally with low-energy excitation is limited in biomedicine and life sciences by its oxygen sensitivity. This hampers the applicability of TF-UC systems in sensors, imaging, optogenetics and drug release. Despite the advances in improving the oxygen tolerability of TF-UC systems, the evaluation of oxygen tolerability is based on comparing the performance at completely deoxygenated (0% oxygen) and ambient (20-21%) conditions, leaving the physiological oxygen levels (0.3-13.5%) neglected. This oversight is not deliberate and is only the result of the lack of simple and predictable methods to obtain and maintain these physiological oxygen levels in an optical setup. Herein, we demonstrate the use of microfluidic chips made of oxygen depleting materials to study the oxygen tolerability of four different micellar nanocarriers made of FDA-approved materials with various oxygen scavenging capabilities by screening their TF-UC performance over physiological oxygen levels. All nanocarriers were capable of efficient TF-UC even in ambient conditions. However, utilizing oxygen scavengers in the oil phase of the nanocarrier improves the oxygen tolerability considerably. For example, at the mean tumour oxygen level (1.4%), nanocarriers made of surfactants and oil phase both capable of oxygen scavenging retained remarkably 80% of their TF-UC emission. This microfluidic concept enables faster, simpler and more realistic evaluation of, not only TF-UC, but any micro or nanoscale oxygen-sensitive system and facilitates their development and implementation in biomedical and life science applications.Peer reviewe
The material-enabled oxygen control in thiol-ene microfluidic channels and its feasibility for subcellular drug metabolism assays under hypoxia in vitro
Tissue oxygen levels are known to be critical to regulation of many cellular processes, including the hepatic metabolism of therapeutic drugs, but its impact is often ignored in in vitro assays. In this study, the material-induced oxygen scavenging property of off-stoichiometric thiol-enes (OSTE) was exploited to create physiologically relevant oxygen concentrations in microfluidic immobilized enzyme reactors (IMERs) incorporating human liver microsomes. This could facilitate rapid screening of, for instance, toxic drug metabolites possibly produced in hypoxic conditions typical for many liver injuries. The mechanism of OSTE-induced oxygen scavenging was examined in depth to enable precise adjustment of the on-chip oxygen concentration with the help of microfluidic flow. The oxygen scavenging rate of OSTE was shown to depend on the type and the amount of the thiol monomer used in the bulk composition, and the surface-to-volume ratio of the chip design, but not on the physical or mechanical properties of the bulk. Our data suggest that oxygen scavenging takes place at the polymer-liquid interface, likely via oxidative reactions of the excess thiol monomers released from the bulk with molecular oxygen. Based on the kinetic constants governing the oxygen scavenging rate in OSTE microchannels, a microfluidic device comprising monolithically integrated oxygen depletion and IMER units was designed and its performance validated with the help of oxygen-dependent metabolism of an antiretroviral drug, zidovudine, which yields a cytotoxic metabolite under hypoxic conditions.Peer reviewe
Pharmacokinetic Simulations of Intravitreal Biologicals : Aspects of Drug Delivery to the Posterior and Anterior Segments
Biologicals are important ocular drugs that are be delivered using monthly and bimonthly intravitreal injections to treat retinal diseases, such as age-related macular degeneration. Long acting delivery systems are needed for prolongation of their dosing interval. Intravitreal biologicals are eliminated from the eye via the aqueous humor outflow. Thus, the anterior and posterior segments are exposed to the drug. We utilized a kinetic simulation model to estimate protein drug concentrations in the vitreous and aqueous humor after bolus injection and controlled release administration to the vitreous. The simulations predicted accurately the experimental levels of 5 biologicals in the vitreous and aqueous humor. The good match between the simulations and experimental data demonstrated almost complete anterior segment bioavailability, and major dose sparing with ocular controlled release systems. Overall, the model is a useful tool in the design of intraocular delivery of biologicals.Biologicals are important ocular drugs that are be delivered using monthly and bimonthly intravitreal injections to treat retinal diseases, such as age-related macular degeneration. Long acting delivery systems are needed for prolongation of their dosing interval. Intravitreal biologicals are eliminated from the eye via the aqueous humor outflow. Thus, the anterior and posterior segments are exposed to the drug. We utilized a kinetic simulation model to estimate protein drug concentrations in the vitreous and aqueous humor after bolus injection and controlled release administration to the vitreous. The simulations predicted accurately the experimental levels of 5 biologicals in the vitreous and aqueous humor. The good match between the simulations and experimental data demonstrated almost complete anterior segment bioavailability, and major dose sparing with ocular controlled release systems. Overall, the model is a useful tool in the design of intraocular delivery of biologicals.Peer reviewe
Drug glucuronidation assays on human liver microsomes immobilized on microfluidic flow-through reactors
UDP-glucuronosyltransferases (UGTs), located in the endoplasmic reticulum of liver cells, are an important family of enzymes, responsible for the biotransformation of several endogenous and exogenous chemicals, including therapeutic drugs. However, the phenomenon of 'latency', i.e., full UGT activity revealed by disruption of the microsomal membrane, poses substantial challenges for predicting drug clearance based on in vitro glucuronidation assays. This work introduces a microfluidic reactor design comprising immobilized human liver microsomes to facilitate the study of UGT-mediated drug clearance under flow-through conditions. The performance of the microreactor is characterized using glucuronidation of 8-hydroxyquinoline (via multiple UGTs) and zidovudine (via UGT2B7) as the model reactions. With the help of alamethicin and albumin effects, we show that conducting UGT metabolism assays under flow conditions facilitates in-depth mechanistic studies, which may also shed light on UGT latency.Peer reviewe
Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes
This work describes a new nanotechnology-based immobilization strategy for cytochrome P450s (CYPs), the major class of drug metabolizing enzymes. Immobilization of CYPs on solid supports provides a significant leap forward compared with soluble enzyme assays by enabling the implementation of through-flow microreactors for, for example, determination of time-dependent inhibition. Immobilization of the complex CYP membrane-protein system is however particularly challenging as the preservation of the authentic enzyme kinetic parameters requires the full complexity of the lipid environment. The developed strategy is based on the spontaneous fusion of biotinylated fusogenic liposomes with lipid bilayers to facilitate the gentle biotinylation of human liver microsomes that incorporate all main natural CYP isoforms. The same process is also feasible for the biotinylation of recombinant CYPs expressed in insect cells, same as any membrane-bound enzymes in principle. As a result, CYPs could be immobilized on streptavidin-functionalized surfaces, both those of commercial magnetic beads and customized microfluidic arrays, so that the enzyme kinetic parameters remain unchanged, unlike in previously reported immobilization approaches that often suffer from restricted substrate diffusion to the enzyme's active site and steric hindrances. The specificity and robustness of the functionalization method of customized microfluidic CYP assays are also carefully examined.Peer reviewe