Abstract Drug metabolite profiling and identification studies are nowadays regularly conducted with liquid chromatography (LC) coupled with mass spectrometry (MS) as an analytical tool. The speed, selectivity and sensitivity of modern LC–MS instruments have been significantly increased in recent years. Especially the use of ultra-high-performance LC (UHPLC) in combination with a modern high-resolution MS instrument offers high full scan detection sensitivity, mass accuracy and the detection of both expected and unexpected metabolites in a single LC–MS run. The present study showed that no single LC–MS conditions were suitable for the analysis of a large group of structurally diverse compounds. The testing of optimum conditions for each individual compound led to more high-quality data when chromatographic retention behavior and mass spectrometric ionization efficiency for in vitro metabolite profiling were considered. The developed LC–MS methods were applicable for measuring both the disappearance of the parent compound and the formation of metabolites. Tentative metabolite identification was based on the measured accurate mass time-of-flight (TOF) MS data. In the second part, a rapid and sensitive assay was designed and built for the trapping, screening and characterization of reactive metabolites in vitro. In total, 78 trapped reactive metabolite conjugates were detected and identified based on accurate mass data using 12 structurally different test compounds. The majority of the detected conjugates were reported for the first time. Amine-containing compounds, that formed methylated and cyanide-trapped products after CYP-mediated reaction steps in human liver microsomal (HLM) incubations, were studied further. The observed methylated cyano conjugates were shown to be experimental artifacts, i.e., metabonates. The study also describes the use of traditional high-performance LC (HPLC) and the more modern UHPLC coupled to time-of-flight, triple quadrupole and hybrid linear ion trap mass spectrometers in drug metabolism studies, and reviews on how to choose the most suitable LC–MS system for metabolite profiling purposes in drug discovery and early drug development.

Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on nykyaikana yleisesti käytetty analyysimenetelmä lääkeaineiden aineenvaihduntatuotteiden (metaboliittien) havaitsemisessa ja tunnistamisessa. Modernien LC–MS -laitteiden nopeus, selektiivisyys ja herkkyys ovat merkittävästi parantuneet viime vuosina. Käytettäessä ultrakorkean suorituskyvyn nestekromatografia (UHPLC) yhdessä nykyaikaisen korkean massaresoluution MS-laitteen kanssa on mahdollista havaita kaikki sekä odotetut että odottamattomat metaboliitit yhdellä kertaa. Tutkimalla suurta joukkoa rakenteellisesti erilaisia yhdisteitä voitiin todeta, että yksittäiselle yhdisteelle optimoidut mittausolosuhteet johtivat korkealaatuisempaan dataan kuin yleiset ei-optimoidut olosuhteet, kun arvioitiin sekä kromatografista piikin profiilia ja pidättymistä että…

Abstract Reactive metabolites are believed to be responsible for rare but serious idiosyncratic adverse drug reactions (IADRs) that have led to the withdrawal of numerous drugs from the market. This has resulted in major harm to patients, economic losses for the pharmaceutical companies and represents a serious problem in drug development. Reactive metabolites can be studied by trapping them with suitable nucleophiles, most commonly with glutathione. The glutathione conjugates formed in these reactions can be analyzed with liquid chromatography mass spectrometry (LC/MS) techniques. In this study, new in vitro methods for the detection and analysis of reactive metabolites were developed. The suitability for reactive metabolite screening of different enzyme sources commonly used in vitro were compared. It was found that sub-cellular fractions yielded significantly larger amounts of glutathione-trapped reactive metabolites as compared to the amounts obtained from intact hepatocytes. Additionally, different metabolites were detected in some cases. Biomimetic metalloporphyrin catalysts were tested for their ability to produce larger amounts of glutathione-trapped metabolites relative to liver S9 fraction incubations. An increase in reactive metabolite production was observed with biomimetic models, but not all of the metabolites produced by liver S9 were observed. The glutathione conjugates of pulegone and of its metabolite menthofuran were analyzed with LC/MS/MS, and the fragmentation spectra of N- and S-/N- di-linked glutathione conjugate were interpreted in detail for the first time. These results will enable more efficient screening of reactive metabolites of furan-containing compounds. Acyl glucuronides are metabolites produced from carboxylic acid-containing compounds and can be reactive. A good correlation was found between the acyl migration half-life and the tendency of a drug to cause IADRs. The carboxylic moiety can also be metabolized to yield acyl coenzyme A (CoA) conjugates that may be more reactive than their corresponding acyl glucuronides. The formation of CoA conjugates and additional conjugates formed from them was found to be more likely with drugs that cause IADRs.

Tiivistelmä Reaktiivisten metaboliittien uskotaan olevan syypää tietyntyyppisiin harvinaisiin, mutta vakaviin idiosynkraattisiin lääkehaittavaikutuksiin, jotka ovat johtaneet useiden lääkeaineiden poistamiseen markkinoilta. Ne ovat aiheuttaneet merkittäviä haittoja potilaille, tappioita lääkeyhtiöille ja ovat vakava ongelma lääkekehityksessä. Reaktiivisia metaboliitteja voidaan tutkia vangitsemalla niitä sopivilla nukleofiileillä, yleisimmin glutationilla. Muodostuneet glutationikonjugaatit voidaan sitten analysoida nestekromatografia / massaspektrometrisin tekniikoin. Tässä tutkimuksessa kehitettiin uusia in vitro tapoja havaita ja analysoida reaktiivisia metaboliitteja. Tavallisimmin käytettyjen entsyymilähteiden soveltuvuutta testattiin reaktiivisten metaboliittien seulontaan. Solufraktioiden havaittiin tuottavan huomattavasti…

Abstract Liquid chromatography (LC) combined with mass spectrometry (MS) is a powerful tool for qualitative and quantitative analytics of organic molecules from various matrices, and the use of this hyphenated technique is very common in bioanalytical laboratories. In this study, LC/MS methods and the required sample preparation applications were developed for plant flavonoid and drug metabolism studies. The main focus was in developing methods to be used during cytochrome P450 (CYP) -specific drug interaction studies. Traditional high performance liquid chromatography (HPLC) and new, more efficient and faster ultra-performance liquid chromatography (UPLC) were utilized together with time-of-flight (TOF) and triple quadrupole (QqQ) mass spectrometry. In the flavonoid study, collision-induced radical cleavage of flavonoid glycosides was tested and observed to be a suitable tool for the structure elucidation of the 15 flavonol glycosides extracted from the medicinal plant Rhodiola rosea. Ten of these glycosides were previously unreported in the plant. Several unreported in vivo bupropion metabolites were identified from human urine when developing the method for the new and more extensive in vitro and in vivo N-in-one interaction cocktail assays. The qualified analysis methods developed here enable faster analysis for the N-in-one cocktail assays, in turn enabling a more efficient screening of drugs that affect CYP-enzyme activities. In the case of the human in vitro cocktail assay, fourteen compounds were analyzed using a single LC/MS/MS run. The method has proven to be very reliable and has been used in several interaction studies utilizing different sample matrices. The in vivo cocktail assay that was developed enables totally non-invasive sample collection from the patients, the urine sample being sufficient for the UPLC/MS/MS analysis of all target compounds. The last part of the study consisted of developing a specific and very sensitive UPLC/MS/MS method for the analysis of one of the in vivo cocktail analytes, the antidiabetic drug repaglinide, from human placenta perfusates.

Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on tehokas työväline kvalitatiivisessa ja kvantitatiivisessa analytiikassa, ja tätä tekniikkaa käytetään erityisesti bioalan laboratorioissa. Tässä väitöskirjatyössä kehitettiin ja sovellettiin LC/MS- ja näytteenkäsittelymenetelmiä kasvien flavonoidimetabolian ja lääkeaineiden metaboliatuotteiden tutkimukseen keskittyen erityisesti sytokromi P450 (CYP) -entsyymispesifisten lääkeaineiden interaktiotutkimuksiin tarvittaviin menetelmiin. Työssä hyödynnettiin perinteistä korkean erotuskyvyn nestekromatografiaa (HPLC) ja uutta, suorituskyvyltään vielä tehokkaampaa ja nopeampaa nestekromatografiaa (UPLC) yhdessä lentoaika- (TOF) ja kolmoiskvadrupolimassaspektrometrian (QqQ) kanssa. Tutkimustyön flavonoidimetaboliaan keskittyneessä osuudessa havaittiin törmäyksen aiheuttaman (CID) radikaalipilkkoutumisen soveltuvan lääkinnällisenä kasvina käytetystä ruusujuuresta…

Abstract Master data describes business objects that are shared across an entire enterprise. Master data is a single source of information that should be used across the IT systems and business processes without changing. Definitions and understanding of common data and how well it is understood forms the basis for understanding the master data. The main objective of this study is to clarify how one product data should be understood and defined and to identify the main challenges and the best practices for managing the one product data for business processes. This study approaches one product data for integrated business processes from several perspectives by focusing on one product master data, data ownership, and the importance of a governance model for managing the master data. The means also to determine business value of master data and to ensure that a company’s success in reaching this business value is analysed. The findings of this study reveal the need for balance between business processes, data, and IT systems. The study indicates that a governance model is necessary in conjunction with business processes, data, and IT systems to ensure that an adequate foundation is created for one product data. One product data is the sum of product-related business data and one product master data. One product master data is the “DNA” of a product that is created by the product portfolio management process and is stored and controlled by a Product Lifecycle Management IT-system that updates the receiving systems in business processes with the common product data. One product data forms the basis for integrated business processes. In the product life cycle context, this means that data must be in place from the new product development phase to the maintenance phase, as well as across sales processes, supply chains, and care/service processes. Discontinuous data is harmful as it causes extra costs in management and slows down data analysis, as well as affects the reaction speed around changes on the business side. New business opportunities such as digitalisation may become very difficult if centralised one product data is not in place. It is important to keep in mind that if data integrity and quality are not in place in a company, adding new business models might be very challenging.

Tiivistelmä Master data on informaatiota, joka on määritelty yksiselitteisesti ja sitä käytetään muuttumattomana ylitse eri IT- järjestelmien ja -prosessien. Datamäärityksillä tuetaan liiketoiminnan prosesseja. Datan määritelmät ja yleinen datan ymmärtämisen taso yrityksessä ovat tärkeitä elementtejä, muodostaen pohjan Master data -käsitteelle. Tämän tutkimuksen päätarkoituksena on selkiyttää kuinka yksiselitteinen tuotetieto tulisi ymmärtää ja määritellä. Samalla identifioidaan suurimmat haasteet ja parhaat käytänteet yhdenmukaisen tuotetiedon hallinnalle. Tutkimuksessa keskitytään yhtenäisen master datan käsitteistön, datan omistajuuden, sekä hallinnointimallin tärkeiden näkökulmien kautta kokonaisuuden ymmärtämiseen useista eri…

Abstract Bringing new products faster to the market is increasingly critical. To reach this goal, companies have been improving their product development practices and processes. Rapid product development (RaDe) is a type of incremental product development model, in which new sales items are created by redesigning existing products quickly and inexpensively. This study examines the RaDe challenges and enablers, particularly the ones from the perspective of supply management (SM) and product portfolio management (PPM). The main objective of this study is to recognise and describe the significance of mature SM for RaDe, resulting in successful and efficient product ramp-ups and portfolio renewal. The present study follows a qualitative research approach, which consists of reviewing the literature and analysing the relevant practices and current process settings of several companies representing diverse business areas and industry types. The principal results of this study involve acknowledging and describing the role of mature SM and organised PPM for effective RaDe implementation and product portfolio renewal. The study examines the mature SM from the perspective of 1) the definition of the SM objectives; 2) the alignment of the list of recommended products, buy-items and suppliers; 3) the creation of the product design guidelines, targets and metrics from the SM perspective; and 4) the establishment of the supply capability creation process. The main implications of the present study highlight the importance of the SM maturity that can support managers in RaDe related planning and implementation activities. The originality of the study resides in the emphasis of the SM significance in terms of different aspects that are beneficial to RaDe implementation and product portfolio renewal.

Tiivistelmä Uusien tuotteiden tuominen markkinoille nopeasti on enenevissä määrin tärkeää yrityksille. Tämän saavuttamiseksi yritykset ovat pyrkineet parantamaan tuotekehitystoimintojaan ja -prosessejaan. Nopea tuotekehitys (RaDe) on malli inkrementaaliseen tuotekehitykseen, missä myyntinimikkeitä luodaan uudelleen suunnittelemalla jo olemassa olevia tuotteita nopeasti ja halvalla. Tämä tutkimus tarkastelee RaDe -haasteita ja mahdollistajia, erityisesti hankintatoimen johtamisen (SM) ja tuoteportfoliohallinnan (PPM) näkökulmasta. Tutkimuksen ensisijaisena tavoitteena on tunnistaa ja kuvata kypsän hankintatoimen johtamisen ja nopean tuotekehityksen merkittävyys niin onnistuneen kuin tehokkaan markkinoille tuonnin sekä tuoteportfolion uudistamisen mahdollistajana. Laadullinen tutkimus tarkastelee monipuolisia liiketoiminta-alueita ja teollisuusaloja, koostuen sekä kirjallisuuskatsauksesta että useiden eri yritysten niille relevanttien toimintojen ja prosessien analysoinnista. Keskeiset tutkimuksen tulokset osoittavat ja selittävät kypsän hankintatoimen johtamisen ja organisoidun tuoteportfoliohallinnan roolia nopean tuotekehityksen tehokkaassa toteutuksessa sekä tuoteportfolion uudistamisessa. Kypsää hankintatoimen johtamista…

Abstract Companies are finding new ways to do business to survive in today’s competition. To meet their business objectives, companies can gain an access to resources and assets by cooperating with other companies. Co-marketing is a form of interorganisational cooperation in which companies combine their marketing resources to gain benefits they would not gain by themselves. However, the lack of understanding over the companies’ own product offerings is potentially hindering the success of co-marketing activities. Productisation over vertical product structure levels and product lifecycle phases may help in gaining the needed understanding. The main objective of this dissertation is to clarify the role of vertical productisation over product lifecycle in co-marketing through a joint commercial product portfolio. Co-marketing through a joint commercial product portfolio is introduced as a novel form of co-marketing. Qualitative research was conducted to study companies’ current practices on productisation and product portfolio management, clarify the role of productisation as a precondition for co-marketing through a joint commercial product portfolio, and construct an approach to productise a joint commercial product portfolio. The results indicate that companies should productise their independent offerings vertically over product structure levels and horizontally over lifecycle phases to gain an understanding over the sellable and deliverable offering in different product lifecycle phases. The vertical and horizontal productisation dimensions should be utilised also in product portfolio management target-setting to help in making decisions on products. Having a clear and up-to-date understanding of the companies’ current independent product portfolios acts as a prerequisite for maintaining an up-to-date joint commercial product portfolio. A common product structure logic should be utilised to productise a joint commercial product portfolio.

Tiivistelmä Yritykset etsivät uusia tapoja selvitäkseen nykyajan kilpaillussa liiketoimintaympäristössä. Jotta yritykset saavuttaisivat liiketoiminnalle asettamansa tavoitteet, ne voivat hankkia pääsyn resursseihin ja muuhun pääomaan yhteistyön kautta. Yksi yhteistyön muoto on yhteismarkkinointi, jossa yritykset yhdistävät markkinointiresurssinsa saavuttaakseen suurempia hyötyjä kuin mitä kukin yritys voisi saavuttaa toimimalla yksin. Yritysten ymmärrys omista tuotetarjoamistaan ei kuitenkaan ole riittävällä tasolla, mikä voi haitata yhteismarkkinoinnin tavoitteiden saavuttamista. Tuotteistaminen vertikaalisten tuoterakennetasojen ja tuotteen elinkaarivaiheiden yli voi edesauttaa riittävän ymmärryksen saavuttamisessa. Tämän väitöstutkimuksen päätavoite on selventää yli tuotteen elinkaaren tapahtuvan vertikaalisen tuotteistamisen roolia yhteisen kaupallisen tuoteportfolion kautta tapahtuvassa markkinointiyhteistyössä. Markkinointi yhteisen kaupallisen tuoteportfolion kautta esitellään uudenlaisena yhteismarkkinoinnin muotona. Tutkimuksessa käytettiin laadullisia menetelmiä…

Abstract Liquid chromatography (LC) combined with mass spectrometry (MS) is one of the most widely used techniques in modern analytical laboratories. Remarkable developments during the two previous decades in both techniques has made LC-MS the method of choice in various environmental, pharmaceutical and biochemical laboratories due to selectivity, sensitivity and versatility. The main focuses in this study were to develop new LC-MS methods to identify and quantify phenolic secondary metabolites in bilberry, lingonberry and hybrid bilberry, to study the biosynthesis of the main secondary metabolites (hypericin and hyperforin and their derivatives) in St John’s wort (SJW) both in vitro and in vivo (in plant), to identify in vitro metabolites of hyperforin in human liver microsomes and to identify the cytochrome P450 (CYP) enzymes responsible for their formation. Both high-performance liquid chromatography (HPLC) and ultra high-performance liquid chromatography (U-HPLC) in combination with time-of-flight (TOF) and triple quadrupole (QqQ) mass spectrometry were used in this study. Identification of 52 phenolic compounds from the leaves of bilberry, lingonberry and hybrid bilberry was accomplished. In total, seven of the identified compounds were reported for the first time in Vaccinium plants and several other compounds were reported for the first time in the studied plants. Incorporation of valine and isoleucine into acyl side chains of phloroglucinols (hyperforin and adhyperforin) via biosynthesis in shoot cultures of SJW was confirmed by using isotopically labeled amino acids and HPLC-MS/MS. Also, 29 biosynthetic in vitro products for HpPKS2 enzyme originating from SJW were identified based on accurate mass data. The metabolism of hyperforin was studied in human liver microsomes (HLM) for the first time. 57 metabolites for hyperforin were identified in the incubations with HLMs, using a substrate concentration of 1 μM. The phase I metabolism of hyperforin was suggested to rely mainly on CYP3A4 and on CYP2C family.

Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on yksi eniten käytetyistä analyysimenetelmistä nykyaikaisissa analytiikkalaboratorioissa. Viimeisten parin vuosikymmenen aikana LC-MS -laitteet ovat kehittyneet merkittävästi, ja nykyään LC-MS onkin paras menetelmä moniin ympäristö-, lääkeaine- ja biokemiallisiin laboratorioihin sen selektiivisyyden, herkkyyden ja monipuolisuuden vuoksi. Tässä väitöskirjassa kehitettiin uusia LC-MS –menetelmiä mustikan, puolukan ja mustikkapuolukan fenolisten sekundäärimetaboliittien tunnistamiseksi ja kvantitoimiseksi, mäkikuisman pääasiallisten sekundäärimetaboliittien (hyperisiini, hyperforiini ja niiden johdannaiset) tutkimiseksi in vitro- ja kasvinäytteistä sekä hyperforiinin aineenvaihduntatuotteiden tunnistamiseksi ja niitä muodostavien sytokromi P450 (CYP) entsyymien tunnistamiseksi ihmisen maksamikrosomeissa in vitro -menetelmin. Tässä työssä käytettiin sekä korkean erotuskyvyn nestekromatografia (HPLC) että ultra-korkean…